scholarly journals Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun’s Nuclear Translocation and Activity

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1806
Author(s):  
Melanie Kappelmann-Fenzl ◽  
Silke Kuphal ◽  
Rosemarie Krupar ◽  
Dirk Schadendorf ◽  
Viktor Umansky ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Stability ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
Microtubule Network ◽  
Localization Sequence ◽  
Dynamic Structures ◽  
Main Regulator ◽  
Factor C

Microtubules are highly dynamic structures, which consist of α- and β-tubulin heterodimers. They are essential for a number of cellular processes, including intracellular trafficking and mitosis. Tubulin-binding chemotherapeutics are used to treat different types of tumors, including malignant melanoma. The transcription factor c-Jun is a central driver of melanoma development and progression. Here, we identify the microtubule network as a main regulator of c-Jun activity. Monomeric α-tubulin fosters c-Jun protein stability by protein–protein interaction. In addition, this complex formation is necessary for c-Jun’s nuclear localization sequence binding to importin 13, and consequent nuclear import and activity of c-Jun. A reduction in monomeric α-tubulin levels by treatment with the chemotherapeutic paclitaxel resulted in a decline in the nuclear accumulation of c-Jun in melanoma cells in an experimental murine model and in patients’ tissues. These findings add important knowledge to the mechanism of the action of microtubule-targeting drugs and indicate the newly discovered regulation of c-Jun by the microtubule cytoskeleton as a novel therapeutic target for melanoma and potentially also other types of cancer.

Characterization of the nuclear translocation of acidic fibroblast growth factor

1993 ◽  
Vol 104 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Y. Cao ◽  
M. Ekstrom ◽  
R.F. Pettersson
Keyword(s):  
Cell Nucleus ◽  
Nuclear Translocation ◽  
Expression System ◽  
Subcellular Fractionation ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
Acidic Fibroblast Growth Factor ◽  
Rabbit Polyclonal Antibody ◽  
Localization Sequence ◽  
Virus Expression

The subcellular localization of human acidic FGF (aFGF; FGF-1) expressed to high levels by using a bacteriophage T7 RNA polymerase-driven vaccinia virus expression system was studied in BHK21 and HeLa cells. Acidic FGF was detected by immunoblotting or immunofluorescence using an affinity-purified rabbit polyclonal antibody. The nuclei of most transfected cells, but not nuclei of control cells, were strongly immunoreactive. The nuclear accumulation of aFGF was confirmed by subcellular fractionation and immunoblotting, indicating that about 50% of the expressed protein was located in the nuclei at 12 h after transfection. It has previously been reported that a putative N-terminal nuclear localization sequence (NLS) in aFGF is required for full mitogenic activity (Imamura et al., Science 249, 1567–1570, 1990). We found that deletion of the first 27 residues including the putative NLS did not prevent the nuclear translocation of aFGF in either cell type. This observation suggests that the putative NLS sequence is not essential for targeting aFGF to the cell nucleus. To analyze further the mechanism of nuclear import, purified aFGF was microinjected into the cytoplasm of growing BHK21 cells under various conditions. In chilled (4 degrees C) or ATP-depleted cells, the injected aFGF entered the nucleus with similar efficiency to that in control cells at 37 degrees C. This suggests that aFGF, which has a molecular mass of only 16,500, enters the cell nucleus by free diffusion, and possibly becomes trapped by binding to some nuclear structures. When added exogenously to growing BHK21 cells, aFGF was not localized to the nucleus. Instead, a punctate staining pattern in the cytosol was observed, reminiscent of that in the endosomal-lysosomal compartments. In addition, a diffuse extracellular surface-staining was evident. This result demonstrates that receptor-mediated endocytosis of aFGF does not result in its translocation to the nucleus, as has been reported for basic FGF.

The sal3+ Gene Encodes an Importin-β Implicated in the Nuclear Import of Cdc25 in Schizosaccharomyces pombe

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 689-703 ◽  
Author(s):  
Gordon Chua ◽  
Carol Lingner ◽  
Corey Frazer ◽  
Paul G Young
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
G1 Arrest ◽  
Mitotic Entry ◽  
Localization Sequence ◽  
Mutant Phenotypes

Abstract In Schizosaccharomyces pombe, the nuclear accumulation of Cdc25 peaks in G2 and is necessary for the proper timing of mitotic entry. Here, we identify the sal3+ gene product as an importin-β homolog that participates in the nuclear import of Cdc25. Loss of sal3+ results in a cell cycle delay, failure to undergo G1 arrest under nitrogen-starvation conditions, and mislocalization of Cdc25 to the cytosol. Fusion of an exogenous classical nuclear localization sequence (cNLS) to Cdc25 restores its nuclear accumulation in a sal3 disruptant and suppresses the sal3 mutant phenotypes. In addition, we show that enhanced nuclear localization of Cdc25 at endogenous levels of expression advances the onset of mitosis. These results demonstrate that the nuclear translocation of Cdc25 is important for the timing of mitotic entry and that Sal3 plays an important role in this process.

The COOH-terminal nuclear localization sequence of interferon gamma regulates STAT1 alpha nuclear translocation at an intracellular site

2000 ◽  
Vol 113 (15) ◽  
pp. 2771-2781
Author(s):  
P.S. Subramaniam ◽  
J. Larkin ◽  
M.G. Mujtaba ◽  
M.R. Walter ◽  
H.M. Johnson
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Biological Activities ◽  
Nuclear Localization Sequence ◽  
Receptor Complex ◽  
Ifn Gamma ◽  
High Affinity ◽  
Gamma Receptor ◽  
Localization Sequence

We have recently shown that the nuclear localization of IFN gamma is mediated by a polybasic nuclear localization sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFN gamma, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the nuclear translocation of the transcription factor STAT1 alpha activated by IFN gamma. Treatment of IFN gamma with antibodies to the C-terminal region (95–133) containing the NLS blocked the induction of STAT1 alpha nuclear translocation. The antibodies had no effect on nuclear translocation of STAT1 alpha in IFN gamma treated cells. A deletion mutant of human IFN gamma, IFN gamma (1–123), which is devoid of the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFN gamma receptor complex on cells with a K(d) similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFN gamma(1–123) to initiate the nuclear translocation of STAT1 alpha, which is required for the biological activities of IFN gamma following binding to the IFN gamma receptor complex. Thus, the NLS region appears to contribute minimally to extracellular high-affinity receptor-ligand binding, yet exerts a strong functional role in STAT1 alpha nuclear localization. A high-affinity site for the interaction of the C-terminal NLS domain of IFN gamma with a K(d) approx. 3 × 10(−8) M(−1) has been described by previous studies on the intracellular cytoplasmic domain of the IFN gamma receptor alpha-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C-terminal NLS domain of IFN gamma into the cytoplasm of cells before treatment of cells with IFN gamma. These intracellular antibodies specifically blocked the nuclear translocation of STAT1 alpha following the subsequent treatment of these cells extracellularly with IFN gamma. These data show that the NLS domain of IFN gamma interacts at an intracellular site to regulate STAT1 alpha nuclear import. A C-terminal peptide of murine IFN gamma, IFN gamma(95–133), that contains the NLS motif, induced nuclear translocation of STAT1 alpha when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1 alpha nuclear translocation. In cells activated with IFN gamma, IFN gamma was found to as part of a complex that contained STAT1 alpha and the importin-alpha analog Npi-1, which mediates STAT1 alpha nuclear import. The tyrosine phosphorylation of STAT1 alpha, the formation of the complex IFN gamma/Npi-1/STAT1 alpha complex and the subsequent nuclear translocation of STAT1 alpha were all found to be dependent on the presence of the IFN gamma NLS. Thus, the NLS of IFN gamma functions intracellularly to directly regulate the activation and ultimate nuclear translocation STAT1 alpha.

scholarly journals PlexinB1 Promotes Nuclear Translocation of the Glucocorticoid Receptor

Cells ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 3
Author(s):  
Magali Williamson ◽  
Ritu Garg ◽  
Claire M. Wells
Keyword(s):  
Prostate Cancer ◽  
Glucocorticoid Receptor ◽  
Cancer Cells ◽  
Androgen Deprivation Therapy ◽  
Late Stage ◽  
Nuclear Translocation ◽  
Androgen Deprivation ◽  
Nuclear Localization Sequence ◽  
Prostate Cancer Cells ◽  
Localization Sequence

Androgen receptor (AR) and glucocorticoid receptor (GR) are nuclear receptors whose function depends on their entry into the nucleus where they activate transcription of an overlapping set of genes. Both AR and GR have a role in resistance to androgen deprivation therapy (ADT), the mainstay of treatment for late stage prostate cancer. PlexinB1, a receptor for semaphorins, has been implicated in various cancers including prostate cancer and has a role in resistance to ADT. We show here that activation of PlexinB1 by Sema4D and Sema3C results in translocation of endogenous GR to the nucleus in prostate cancer cells, and that this effect is dependent on PlexinB1 expression. Sema4D/Sema3C promotes the translocation of GR-GFP to the nucleus and mutation of the nuclear localization sequence (NLS1) of GR abrogates this response. These findings implicate the importin α/β system in the Sema4D/Sema3C-mediated nuclear import of GR. Knockdown of PlexinB1 in prostate cancer cells decreases the levels of glucocorticoid-responsive gene products and antagonizes the decrease in cell motility and cell area of prostate cancer cells upon dexamethasone treatment, demonstrating the functional significance of these findings. These results show that PlexinB1 activation has a role in the trafficking and activation of the nuclear receptor GR and thus may have a role in resistance to androgen deprivation therapy in late stage prostate cancer.

scholarly journals The checkpoint-dependent nuclear accumulation of Rho1p exchange factor Rgf1p is important for tolerance to chronic replication stress

2014 ◽  
Vol 25 (7) ◽  
pp. 1137-1150 ◽  
Author(s):  
Sofía Muñoz ◽  
Elvira Manjón ◽  
Patricia García ◽  
Per Sunnerhagen ◽  
Yolanda Sánchez
Keyword(s):  
Rho Gtpases ◽  
Nuclear Export ◽  
Replication Stress ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
General Interest ◽  
Nucleotide Exchange ◽  
Replication Checkpoint ◽  
Exchange Factor ◽  
Localization Sequence

Guanine nucleotide exchange factors control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is relocalized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence at the N-terminus of Rgf1p, whereas release into the cytoplasm requires two leucine-rich nuclear export sequences at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes nine serine potential phosphorylation Cds1p sites for alanine fails to accumulate in the nucleus in response to replication stress, and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologues of Rgf1p in budding yeast and higher eukaryotes.

scholarly journals SCCRO (DCUN1D1) Promotes Nuclear Translocation and Assembly of the Neddylation E3 Complex

2011 ◽  
Vol 286 (12) ◽  
pp. 10297-10304 ◽  
Author(s):  
Guochang Huang ◽  
Andrew J. Kaufman ◽  
Y. Ramanathan ◽  
Bhuvanesh Singh
Keyword(s):  
Nuclear Localization ◽  
Essential Role ◽  
Nuclear Translocation ◽  
Nuclear Localization Sequence ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Transgenic Expression ◽  
Localization Sequence ◽  
Efficient Transfer

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO−/− mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO−/− mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.

scholarly journals Analysis of mutations in the putative nuclear localization sequence of interleukin-1β

1991 ◽  
Vol 280 (1) ◽  
pp. 111-116 ◽  
Author(s):  
S Grenfell ◽  
N Smithers ◽  
S Witham ◽  
A Shaw ◽  
P Graber ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Interleukin 1 ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
Target Cells ◽  
Wild Type ◽  
Receptor Mediated Endocytosis ◽  
Localization Sequence

Previous studies have shown that, after receptor-mediated endocytosis, interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) are translocated to the nucleus, where they appear to accumulate. It has been suggested that nuclear translocation may be involved in the biological responsiveness of target cells to IL1 stimulation. The human IL1 beta molecule contains a seven-amino-acid sequence (-Pro208-Lys-Lys-Lys-Met-Glu-Lys-) that shows some sequence identity with the nuclear localization sequence of the simian-virus-40 large T-antigen. The effects of point mutations within this putative nuclear localization sequence on IL1 beta binding, receptor-mediated endocytosis and biological activity have been characterized. Mutants M49 (Lys210→Ala), M50 (Lys211→Ala) and M51 (Pro208→Ala) all retained the ability to bind to the IL1 receptor, albeit with lower affinity than the wild-type molecules. However, mutants M49, M50 and M51 showed greater biological potency than wild-type IL1 alpha or IL1 beta, as measured by the induction of IL2 secretion. However, receptor-mediated endocytosis and nuclear accumulation of M50 were comparable with those in the wild-type. These observations suggest that the putative nuclear localization sequence may play an important role in the generation of biological responses to IL1 stimulation, even though it may not influence internalization of the ligand.

scholarly journals Design of Conditionally Active STATs: Insights into STAT Activation and Gene Regulatory Function

1999 ◽  
Vol 19 (4) ◽  
pp. 2913-2920 ◽  
Author(s):  
Lawrence H. Milocco ◽  
Jennifer A. Haslam ◽  
Jonathan Rosen ◽  
H. Martin Seidel
Keyword(s):  
Tyrosine Phosphorylation ◽  
Nuclear Translocation ◽  
Regulatory Function ◽  
Nuclear Localization Sequence ◽  
Wild Type ◽  
Localization Sequence ◽  
Gene Regulatory ◽  
First Time ◽  
Insight Into

ABSTRACT The STAT (signal transducer and activator of transcription) signaling pathway is activated by a large number of cytokines and growth factors. We sought to design a conditionally active STAT that could not only provide insight into basic questions about STAT function but also serve as a powerful tool to determine the precise biological role of STATs. To this end, we have developed a conditionally active STAT by fusing STATs with the ligand-binding domain of the estrogen receptor (ER). We have demonstrated that the resulting STAT-ER chimeras are estrogen-inducible transcription factors that retain the functional and biochemical characteristics of the cognate wild-type STATs. In addition, these tools have allowed us to evaluate separately the contribution of tyrosine phosphorylation and dimerization to STAT function. We have for the first time provided experimental data supporting the model that the only apparent role of STAT tyrosine phosphorylation is to drive dimerization, as dimerization alone is sufficient to unmask a latent STAT nuclear localization sequence and induce nuclear translocation, sequence-specific DNA binding, and transcriptional activity.

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