Importin 7 and Nup358 Promote Nuclear Import of the Protein Component of Human Telomerase

Frohnert, Cornelia; Hutten, Saskia; Wälde, Sarah; Nath, Annegret; Kehlenbach, Ralph H.

doi:10.1371/journal.pone.0088887

Cited by 25 publications

(25 citation statements)

References 51 publications

(83 reference statements)

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“…6B). As a positive control for the method we also confirmed that importin 7 coimmunoprecipitated with hTERT-GFP (data not shown), as this interaction had been reported previously (Frohnert et al, 2014). Finally, we used importin 4 as a negative control, since knockdown of cellular levels of importin 4 had no effect on TRα1 localization.…”

Section: Resultssupporting

confidence: 89%

“…The GFP-glutathione-S-transferase (GST)-GFP (G3) expression vector, G3- A/BD (containing NLS-2) expression plasmid, and G3-Hinge (containing NLS-1) expression plasmid were previously described (Mavinakere et al, 2012). The plasmid hTERT-GFP was a gift from R. H. Kehlenbach (University of Göttingen) and encodes GFP-tagged human telomerase reverse transcriptase (Frohnert et al, 2014). pGST-GFP-NLS was a gift from R. J.G.…”

Section: Methodsmentioning

confidence: 99%

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Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin β1, and adaptor importin α1

Roggero

Zhang

Parente

et al. 2016

Molecular and Cellular Endocrinology

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The thyroid hormone receptor α1 (TRα1) is a nuclear receptor for thyroid hormone that shuttles rapidly between the nucleus and cytoplasm. Our prior studies showed that nuclear import of TRα1 is directed by two nuclear localization signals, one in the N-terminal A/B domain and the other in the hinge domain. Here, we showed using in vitro nuclear import assays that TRα1 nuclear localization is temperature and energy-dependent and can be reconstituted by the addition of cytosol. In HeLa cells expressing green fluorescent protein (GFP)-tagged TRα1, knockdown of importin 7, importin β1 and importin α1 by RNA interference, or treatment with an importin β1-specific inhibitor, significantly reduced nuclear localization of TRα1, while knockdown of other importins had no effect. Coimmunoprecipitation assays confirmed that TRα1 interacts with importin 7, as well as importin β1 and the adapter importin α1, suggesting that TRα1 trafficking into the nucleus is mediated by two distinct pathways.

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Section: Resultssupporting

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin β1, and adaptor importin α1

Roggero

Zhang

Parente

et al. 2016

Molecular and Cellular Endocrinology

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“…Importin 7 has been recently shown to interact with hTERT and to mediate nuclear transport of hTERT (Frohnert et al, 2014). To confirm these results in our system, we examined whether importin 7 binds to the hTERT NLS, and whether its depletion affects the levels and subcellular localization of hTERT and telomerase activity.…”

Section: Discussionsupporting

confidence: 61%

“…Thus, it is of interest to investigate whether phosphorylation of S227 affects the interaction between the hTERT NLS and the import receptors. Recently, importin 7 has been shown to interact with the hTERT NLS and to mediate nuclear transport of hTERT (Frohnert et al, 2014). However, the molecular mechanism regulating the nuclear import of hTERT remains unclear.…”

Section: Introductionmentioning

confidence: 99%

Akt-mediated phosphorylation increases the binding affinity of hTERT for importin α to promote nuclear translocation

Jeong

Kim

Lee

et al. 2015

Journal of Cell Science

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There was an error published in J. Cell Sci. 128, 2287-2301.In Fig. 2, the HA-importin-α3 immunoprecipitation and corresponding Flag-NLS blots were inadvertently duplicated as the HA-importin-α5 immunoprecipitation and corresponding Flag-NLS blots. The two HA-importin-α5 blots have been replaced with the correct images in panel D in the figure shown below. There are no changes to the figure legend, which is accurate. This error does not affect the conclusions of the study.The authors apologise to the readers for any confusion that this error might have caused. Fig. 2. The hTERT NLS interacts physically with importin α. (A) GST-importin-α1, GST-importin-α3, GST-importin-α5 and GST-importin-β1 were affinity purified and incubated with lysates that had been prepared from cells expressing Flag-NLS or Flag-S227A/7A, followed by immunoblotting with anti-Flag antibody. The purified GST fusion proteins were visualized by Coomassie staining and are indicated with arrowheads. Molecular mass makers are shown in kDa. (B) GST-NLS and GST-S227A/7A were purified and incubated with lysates prepared from cells expressing HA-importin-α1, HA-importin-α3, HA-importin-α5 and HA-importin-β1; the HA-tagged proteins were then detected. HA-tagged importin proteins are indicated with arrowheads. The purified GST fusion proteins were visualized by Coomassie staining. (C) MCF7 cells that had been transfected with Flag-NLS and either HA-importin-α proteins or HA-importin-β1 were subjected to immunoprecipitation (IP) with anti-Flag antibody, followed by immunoblotting with anti-HA antibody. HA-tagged importin proteins are indicated with arrowheads. (D) MCF7 cells transfected with Flag-NLS, HA-importin-α proteins and HA-importin-β1 were subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-HA antibody. (E) MCF7 cells were subjected to immunoprecipitation with an anti-hTERT antibody; the immunoprecipitates were then probed for endogenous importin-α proteins and importin-β1. IgG antibody was used as a negative control. In B,C,D, asterisks mark the position of nonspecific immunoglobulin chains. ABSTRACTTelomeres are essential for chromosome integrity and protection, and their maintenance requires the ribonucleoprotein enzyme telomerase. Previously, we have shown that human telomerase reverse transcriptase (hTERT) contains a bipartite nuclear localization signal (NLS; residues 222-240) that is responsible for nuclear import, and that Akt-mediated phosphorylation of residue S227 is important for efficient nuclear import of hTERT. Here, we show that hTERT binds to importin-α proteins through the bipartite NLS and that this heterodimer then forms a complex with importin-β proteins to interact with the nuclear pore complex. Depletion of individual importin-α proteins results in a failure of hTERT nuclear import, and the resulting cytoplasmic hTERT is degraded by ubiquitin-dependent proteolysis. Crystallographic analysis reveals that the bipartite NLS interacts with both the major and minor sites of importin-α...

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“…However, the precise mechanism of hTERT uptake and presentation to CD4 T cells had been poorly studied. Indeed, few studies were focused on hTERT presentation on MHC-II mainly due to the inability to produce recombinant hTERT protein (14)(15)(16). Recently, Kellermann et al (17) described a method to produce recombinant hTERT protein.…”

mentioning

confidence: 99%

Heparan Sulfate Proteoglycans Promote Telomerase Internalization and MHC Class II Presentation on Dendritic Cells

Galaine

Kellermann

Guillaume

et al. 2016

The Journal of Immunology

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Telomerase is a prototype-shared tumor Ag and represents an attractive target for anticancer immunotherapy. We have previously described promiscuous and immunogenic HLA-DR–restricted peptides derived from human telomerase reverse transcriptase (hTERT) and referred as universal cancer peptide (UCP). In nonsmall cell lung cancer, the presence of spontaneous UCP-specific CD4 T cell responses increases the survival of chemotherapy-responding patients. However, the precise mechanisms of hTERT’s uptake, processing, and presentation on MHC-II molecules to stimulate CD4 T cells are poorly understood. In this work, by using well-characterized UCP-specific CD4 T cell clones, we showed that hTERT processing and presentation on MHC-II involve both classical endolysosomal and nonclassical cytosolic pathways. Furthermore, to our knowledge, we demonstrated for the first time that hTERT’s internalization by dendritic cells requires its interaction with surface heparan sulfate proteoglycans. Altogether, our findings provide a novel mechanism of tumor-specific CD4 T cell activation and will be useful for the development of novel cancer immunotherapies that harness CD4 T cells.

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Importin 7 and Nup358 Promote Nuclear Import of the Protein Component of Human Telomerase

Cited by 25 publications

References 51 publications

Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin β1, and adaptor importin α1

Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin β1, and adaptor importin α1

Akt-mediated phosphorylation increases the binding affinity of hTERT for importin α to promote nuclear translocation

Heparan Sulfate Proteoglycans Promote Telomerase Internalization and MHC Class II Presentation on Dendritic Cells

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