scholarly journals SUMOylation of the Polyglutamine Repeat Protein, Ataxin-1, Is Dependent on a Functional Nuclear Localization Signal

2005 ◽  
Vol 280 (23) ◽  
pp. 21942-21948 ◽  
Author(s):  
Brigit E. Riley ◽  
Huda Y. Zoghbi ◽  
Harry T. Orr
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
High Mobility ◽  
Wild Type ◽  
Repeat Protein ◽  
Polyglutamine Repeat ◽  
Lysine Residues ◽  
Localization Signal ◽  
Functional Nuclear Localization Signal ◽  
Polyglutamine Tract

SUMO (small ubiquitin-like modifier) is a member of the ubiquitin family of proteins. SUMO targets include proteins involved in numerous roles including nuclear transport and transcriptional regulation. The previous finding that mutant ataxin-1[82Q] disrupted promyelocytic leukemia (PML) oncogenic domains prompted us to determine whether ataxin-1 disrupts another component of PML oncogenic domains, Sp100 (100-kDa Speckled protein). Similar to the PML protein, mutant ataxin-1[82Q] redistributed Sp100 to mutant ataxin-1[82Q] nuclear inclusions. Based on the ability of PML and Sp100 to be covalently modified by SUMO, we investigated the ability of ataxin-1 to be SUMOylated. SUMO-1 was found to covalently modify the polyglutamine repeat protein ataxin-1. There was a decrease in ataxin-1 SUMOylation in the presence of the expanded polyglutamine tract, ataxin-1[82Q]. The phospho-mutant, ataxin-1[82Q]-S776A, restored SUMO levels to those of wild-type ataxin-1[30Q]. SUMOylation of ataxin-1 was dependent on a functional nuclear localization signal. Ataxin-1 SUMOylation was mapped to at least five lysine residues. Lys16, Lys194 preceding the polyglutamine tract, Lys610/Lys697 in the C-terminal ataxin high mobility group domain, and Lys746 all contribute to ataxin-1 SUMOylation.

Mutations of the SBDS Gene Result in Nuclear Mislocalization.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2034-2034
Author(s):  
Masafumi Yamaguchi ◽  
Kingo Fujimura ◽  
Hanae Toga-Yamaguchi ◽  
Valentina Svetic ◽  
Naoki Okamura ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Exocrine Pancreatic Insufficiency ◽  
Expression Level ◽  
Wild Type ◽  
Nuclear Localization Signals ◽  
Localization Signal ◽  
Domain I

Abstract Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of granulocyte precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol. Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.

scholarly journals Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights

2013 ◽  
Vol 69 (12) ◽  
pp. 2495-2505 ◽  
Author(s):  
Gergely Róna ◽  
Mary Marfori ◽  
Máté Borsos ◽  
Ildikó Scheer ◽  
Enikő Takács ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nucleocytoplasmic Transport ◽  
Wild Type ◽  
Post Translational Modification ◽  
Nuclear Localization Signals ◽  
M Phase ◽  
Importin Α ◽  
Localization Signal

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α–wild-type and the importin-α–hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.

scholarly journals DeSUMOylation of Gli1 by SENP1 Attenuates Sonic Hedgehog Signaling

2017 ◽  
Vol 37 (18) ◽  
Author(s):  
Huaize Liu ◽  
Sen Yan ◽  
Jie Ding ◽  
Ting-Ting Yu ◽  
Steven Y. Cheng
Keyword(s):  
Sonic Hedgehog ◽  
Nuclear Localization ◽  
Posttranslational Modifications ◽  
Nuclear Localization Signal ◽  
Hedgehog Signaling ◽  
Sonic Hedgehog Signaling ◽  
Lysine Residues ◽  
Localization Signal ◽  
Interfering Rna ◽  
Transcriptional Output

ABSTRACT The transcriptional output of the Sonic Hedgehog morphogenic pathway is orchestrated by three Krüppel family transcription factors, Gli1 to -3, which undergo extensive posttranslational modifications, including ubiquitination and SUMOylation. Here, we report that the sentrin-specific peptidase SENP1 is the specific deSUMOylation enzyme for Gli1. We show that SUMOylation stabilizes Gli1 by competing with ubiquitination at conserved lysine residues and that SUMOylated Gli1 is enriched in the nucleus, suggesting that SUMOylation is a nuclear localization signal for Gli1. Finally, we show that small interfering RNA (siRNA)-mediated knockdown of SENP1 augments the ability of Shh to sustain the proliferation of cerebellar granule cell precursors, demonstrating the physiological significance of the negative regulation of Shh signaling by SENP1.

scholarly journals Extracellular vesicles transfer nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander effects

2018 ◽  
Vol 29 (18) ◽  
pp. 2228-2242 ◽  
Author(s):  
Shubhra Rastogi ◽  
Amini Hwang ◽  
Josolyn Chan ◽  
Jean Y. J. Wang
Keyword(s):  
Extracellular Vesicles ◽  
Dna Damage Response ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Oxygen Species ◽  
Wild Type ◽  
Bystander Effects ◽  
Damage Response ◽  
Localization Signal ◽  
Radiation Induced

Ionizing radiation (IR) not only activates DNA damage response (DDR) in irradiated cells but also induces bystander effects (BE) in cells not directly targeted by radiation. How DDR pathways activated in irradiated cells stimulate BE is not well understood. We show here that extracellular vesicles secreted by irradiated cells (EV-IR), but not those from unirradiated controls (EV-C), inhibit colony formation in unirradiated cells by inducing reactive oxygen species (ROS). We found that µEV-IR from Abl nuclear localization signal–mutated ( Abl-µNLS) cells could not induce ROS, but expression of wild-type Abl restored that activity. Because nuclear Abl stimulates miR-34c biogenesis, we measured miR-34c in EV and found that its levels correlated with the ROS-inducing activity of EV. We then showed that EV from miR-34c minigene–transfected, but unirradiated cells induced ROS; and transfection with miR-34c-mimic, without radiation or EV addition, also induced ROS. Furthermore, EV-IR from miR34-family triple-knockout cells could not induce ROS, whereas EV-IR from wild-type cells could cause miR-34c increase and ROS induction in the miR-34 triple-knockout cells. These results establish a novel role for extracellular vesicles in transferring nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander oxidative stress.

scholarly journals A nuclear localization signal binding protein in the nucleolus.

1990 ◽  
Vol 111 (6) ◽  
pp. 2235-2245 ◽  
Author(s):  
U T Meier ◽  
G Blobel
Keyword(s):  
Rat Liver ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Biochemical Characterization ◽  
T Antigen ◽  
Single Amino Acid ◽  
Wild Type ◽  
Western Blots ◽  
Peptide Conjugates ◽  
Localization Signal

We used functional wild-type and mutant synthetic nuclear localization signal peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) were exclusively recognized by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild type, competed less efficiently. The two proteins were extractable from nuclei by either low or high ionic strength buffers. We purified p140 and raised polyclonal antibodies in chicken against the protein excised from polyacrylamide gels. The anti-p140 antibodies were monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of whole cells. Indirect immunofluorescence microscopy on fixed and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a distinct punctate nucleolar staining. Rhodamine-labeled wild-type peptide conjugates also bound to nucleoli in a similar pattern on fixed and permeabilized BRL cells. Based on biochemical characterization, p140 is a novel nucleolar protein. It is possible that p140 shuttles between the nucleolus and the cytoplasm and functions as a nuclear import carrier.

scholarly journals A hydrophobic protein sequence can override a nuclear localization signal independently of protein context.

1991 ◽  
Vol 11 (10) ◽  
pp. 5137-5146 ◽  
Author(s):  
K van Zee ◽  
F Appel ◽  
E Fanning
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Heterologous Proteins ◽  
Wild Type ◽  
Terminal Sequence ◽  
Localization Signal ◽  
Carboxy Terminal

Simian virus 40 T antigen is specifically targeted to the nucleus by the signal Pro-Lys-Lys-128-Lys-Arg-Lys-Val. We have previously described the isolation of a simian virus 40 T-antigen mutant, 676FS, which retains a wild-type nuclear localization signal but fails to accumulate properly in the nucleus and interferes with the nuclear localization of heterologous proteins. Here we report that the hydrophobic carboxy-terminal sequence novel to 676FS T antigen overrides the nuclear localization signal if fused to other proteins, thereby anchoring the proteins in the cytoplasm. We discuss possible mechanisms by which missorting of such a fusion protein could interfere with the nuclear transport of heterologous proteins.

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