Nuclear import of hTERT requires a bipartite nuclear localization signal mediated by Akt phosphorylation

Chung, Jeeyun; Khadka, Prabhat; Chung, In Kwon

doi:10.1242/jcs.099267

Cited by 86 publications

(89 citation statements)

References 60 publications

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“…We have previously identified a bipartite NLS (222-RRRGGSASRSLPLPKRPRR-240) that is responsible for the nuclear import of hTERT (Chung et al, 2012). Mutations in the two clusters of basic amino acids in the bipartite NLS results in reduced nuclear localization of hTERT.…”

Section: A Bipartite Nls Is Essential For Nuclear Import Of Htertmentioning

confidence: 99%

“…Mutations in the two clusters of basic amino acids in the bipartite NLS results in reduced nuclear localization of hTERT. We have also shown that Akt-mediated phosphorylation at S227 is necessary for efficient nuclear translocation of hTERT (Chung et al, 2012). To determine whether nuclear localization of hTERT is affected by different cellular backgrounds, several cell lines -including MCF7, H1299 and U2OS cells -were transfected with Flag-hTERT and various mutant constructs (Flag-7A, Flag-S227A and Flag-S227E, see Fig.…”

Section: A Bipartite Nls Is Essential For Nuclear Import Of Htertmentioning

confidence: 99%

“…We have previously shown that Akt-mediated phosphorylation at S227 is required for efficient nuclear translocation of hTERT (Chung et al, 2012). To explore the molecular mechanism by which phosphorylation of S227 is involved in the nuclear import of hTERT, we used isothermal titration calorimetry (ITC) to determine the binding affinity of importin α1 and importin α5 for an NLS peptide that was phosphorylated at S227 ( phospho-S227) compared with that for wild-type NLS peptide.…”

Section: Nuclear Import Of Htert Is Dependent On Ranmentioning

confidence: 99%

“…We have previously reported that hTERT contains a bipartite NLS (residues 222-240) and that Akt-mediated phosphorylation of residue S227 on hTERT plays an important role in the nuclear translocation of hTERT (Chung et al, 2012). Interestingly, S227 is located between two clusters of basic amino acids in the bipartite NLS.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: A Bipartite Nls Is Essential For Nuclear Import Of Htertmentioning

confidence: 99%

Section: A Bipartite Nls Is Essential For Nuclear Import Of Htertmentioning

confidence: 99%

Section: Nuclear Import Of Htert Is Dependent On Ranmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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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“…(110) TERT is imported from the cytoplasm to the nucleus through Akt-mediated phosphorylation at serine 227 and its interaction with heath shock protein 90 (Hsp90). TERT localization is also regulated by interaction with other proteins such as nuclear factor kappa B (NF-kB) and tumor necrosis factor a (TNF-a) (111). TERT has a mitochondrial targeting signal and, therefore, TERT can enter the mitochondrial matrix where it interacts with mitochondrial DNA and mitochondrial tRNA (112).…”

Section: Dna Damage and Nddmentioning

confidence: 99%

New Insight in The Molecular Mechanisms of Neurodegenerative Disease

2018

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B ACKGROUND:Redox and proteotoxic stress contributes to age-dependent accumulation of dysfunctional mitochondria and protein aggregates, and is associated with neurodegeneration. The free radical theory of aging inspired many studies using reactive species scavengers such as alpha-tocopherol, ascorbate and coenzyme-Q to suppress the initiation of oxidative stress. However, clinical trials have had limited success in the treatment of neurodegenerative diseases (NDDs). CONTENT:The misfolding and aggregation of specific proteins is a seminal occurrence in a remarkable variety of NDDs. In Alzheimer's disease, the two principal aggregating proteins are β-amyloid (Aβ) and tau. The abnormal assemblies formed by conformational variants of these proteins range in size from small oligomers to the characteristic lesions that are visible by optical microscopy, such as senile plaques and neurofibrillary tangles. Pathologic similarities with prion disease suggest that the formation and spread of these proteinaceous lesions might involve a common molecular mechanism, corruptive protein templating. The accumulation of redox modified proteins or organelles cannot be reversed by oxidant intercepting antioxidants and must then be removed by alternative mechanisms. Autophagy serves this essential function in removing damaged or dysfunctional proteins and organelles thus preserving neuronal function and survival.SUMMARY: Senescent cells and their senescenceassociated secretory phenotypes (SASPs) may constitute a novel, understudied, and potentially important contributor to neuro-inflammation and subsequent neurodegeneration. Characterization of cellular senescence in the brain could uncover novel therapeutic targets for the prevention and treatment of chronic age-related NDDs. KEYwORDS

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Fucoidan Induces ROS‐Dependent Apoptosis in 5637 Human Bladder Cancer Cells by Downregulating Telomerase Activity via Inactivation of the PI3K/Akt Signaling Pathway

Han

Lee

Jeong

et al. 2016

Drug Development Research

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Preclinical Research Fucoidan, a sulfated polysaccharide, is a compound found in various species of seaweed that has anti-viral, anti-bacterial, anti-oxidant, anti-inflammatory, and immunomodulatory activities; however, the underlying relationship between apoptosis and anti-telomerase activity has not been investigated. Here, we report that fucoidan-induced apoptosis in 5637 human bladder cancer cells was associated with an increase in the Bax/Bcl-2 ratio, the dissipation of the mitochondrial membrane potential (MMP, Δψm), and cytosolic release of cytochrome c from the mitochondria. Under the same experimental conditions, fucoidan-treatment decreased hTERT (human telomerase reverse transcriptase) expression and the transcription factors, c-myc and Sp1. This was accompanied by decreased telomerase activity. Fucoidan-treatment also suppressed activation of the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling enhanced fucoidan-induced apoptosis and anti-telomerase activity. Meanwhile, fucoidan treatment increased the generation of intracellular ROS, whereas the over-elimination of ROS by N-acetylcysteine, an anti-oxidant, attenuated fucoidan-induced apoptosis, inhibition of hTERT, c-myc, and Sp1 expression, and reversed fucoidan-induced inactivation of the PI3K/Akt signaling pathway. Collectively, these data indicate that the induction of apoptosis and the inhibition of telomerase activity by fucoidan are mediated via ROS-dependent inactivation of the PI3K/Akt pathway. Drug Dev Res 78 : 37-48, 2017. © 2016 Wiley Periodicals, Inc.

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Nuclear import of hTERT requires a bipartite nuclear localization signal mediated by Akt phosphorylation

Cited by 86 publications

References 60 publications

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

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

New Insight in The Molecular Mechanisms of Neurodegenerative Disease

Fucoidan Induces ROS‐Dependent Apoptosis in 5637 Human Bladder Cancer Cells by Downregulating Telomerase Activity via Inactivation of the PI3K/Akt Signaling Pathway

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