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Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

Huang WR, Chiu HC, Liao TL, Chuang KP, Shih WL, Liu HJ - PLoS ONE (2015)

Bottom Line: To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner.The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4.Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan.

ABSTRACT
Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

No MeSH data available.


Related in: MedlinePlus

A rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells as well as examination of AKT required for p21 phosphorylation.(A) To carry out a rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells, cells were transfected with the EGFP-Tpr plasmids for 48 hours, and then either infected with ARV at an MOI of 10 or p17 transfection for 24 hours. Whole cell lysates were collected for Western blot assays. Histone H2A was used as a loading control. The activation and inactivation folds indicated below each lane were normalized against those in mock controls (cell alone). The levels of indicated proteins in the mock control were considered 1-fold. (B) Representative images of vero cells transfected with plasmids overexpressing EGFP-Tpr. Vero cells were transfected with negative controls (mock and vector only) or EGFP-Tpr plasmid for 48 hours and then visualized by immunofluorescence following nuclear DAPI staining. In addition, another set of cells were transfected with EGFP-Tpr for 48 hours and then transfected with p17 for 24 hours. Cells were fixed and processed for immunofluorescence staining of p17. Colocalization of EGFP-Tpr and p17 was observed under a fluorescence microscope. Scale bar: 10 um. (C) To examine whether AKT kinase activity is directly required for p21, we used an Akt DN to inhibit AKT in Vero cells. The levels of p-p21 (T145) in the nucleus were examined in ARV-infected and p17-transfected cells in the presence or absence of an Akt DN. Histone H2A was used as a loading control.
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pone.0133699.g004: A rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells as well as examination of AKT required for p21 phosphorylation.(A) To carry out a rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells, cells were transfected with the EGFP-Tpr plasmids for 48 hours, and then either infected with ARV at an MOI of 10 or p17 transfection for 24 hours. Whole cell lysates were collected for Western blot assays. Histone H2A was used as a loading control. The activation and inactivation folds indicated below each lane were normalized against those in mock controls (cell alone). The levels of indicated proteins in the mock control were considered 1-fold. (B) Representative images of vero cells transfected with plasmids overexpressing EGFP-Tpr. Vero cells were transfected with negative controls (mock and vector only) or EGFP-Tpr plasmid for 48 hours and then visualized by immunofluorescence following nuclear DAPI staining. In addition, another set of cells were transfected with EGFP-Tpr for 48 hours and then transfected with p17 for 24 hours. Cells were fixed and processed for immunofluorescence staining of p17. Colocalization of EGFP-Tpr and p17 was observed under a fluorescence microscope. Scale bar: 10 um. (C) To examine whether AKT kinase activity is directly required for p21, we used an Akt DN to inhibit AKT in Vero cells. The levels of p-p21 (T145) in the nucleus were examined in ARV-infected and p17-transfected cells in the presence or absence of an Akt DN. Histone H2A was used as a loading control.

Mentions: Importantly, the elevation in p-53 (S15), p-p21 (T145), and PTEN levels in the nucleus were also seen not only in Tpr-depleted cells but also in ARV-infected and p17-transfected cells (Fig 3D; S1D Fig). Furthermore, the effects of both p17-transfection and ARV-infection could be abolished by overexpression of EGFP-Tpr (Fig 4A), suggesting that p17 causes the increase in p-53 (S15), p-p21 (T145), and PTEN levels by a downregulation of Tpr. In the present study, representative images of Vero cells transfected with plasmids overexpressing EGFP-Tpr was shown in Fig 4B. Colocalization of EGFP-Tpr and p17 was observed under a fluorescence microscope (Fig 4B). Since both p21 and PTEN are the downstream targets of p53 [44, 45], upregulation of PTEN and p21 in Tpr-depleted cells was due to activation of p53. Our observations were consistent with a previous report suggesting that depletion of Tpr reduced nuclear pore formation and nucleo-cytoplasmic trafficking activities, thereby causing p53 nuclear accumulation, which induced specific downstream target genes of the p53 pathway [9, 46].


Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

Huang WR, Chiu HC, Liao TL, Chuang KP, Shih WL, Liu HJ - PLoS ONE (2015)

A rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells as well as examination of AKT required for p21 phosphorylation.(A) To carry out a rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells, cells were transfected with the EGFP-Tpr plasmids for 48 hours, and then either infected with ARV at an MOI of 10 or p17 transfection for 24 hours. Whole cell lysates were collected for Western blot assays. Histone H2A was used as a loading control. The activation and inactivation folds indicated below each lane were normalized against those in mock controls (cell alone). The levels of indicated proteins in the mock control were considered 1-fold. (B) Representative images of vero cells transfected with plasmids overexpressing EGFP-Tpr. Vero cells were transfected with negative controls (mock and vector only) or EGFP-Tpr plasmid for 48 hours and then visualized by immunofluorescence following nuclear DAPI staining. In addition, another set of cells were transfected with EGFP-Tpr for 48 hours and then transfected with p17 for 24 hours. Cells were fixed and processed for immunofluorescence staining of p17. Colocalization of EGFP-Tpr and p17 was observed under a fluorescence microscope. Scale bar: 10 um. (C) To examine whether AKT kinase activity is directly required for p21, we used an Akt DN to inhibit AKT in Vero cells. The levels of p-p21 (T145) in the nucleus were examined in ARV-infected and p17-transfected cells in the presence or absence of an Akt DN. Histone H2A was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526660&req=5

pone.0133699.g004: A rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells as well as examination of AKT required for p21 phosphorylation.(A) To carry out a rescue experiment by overexpression of EGFP-Tpr in ARV-infected and p17-transfected vero cells, cells were transfected with the EGFP-Tpr plasmids for 48 hours, and then either infected with ARV at an MOI of 10 or p17 transfection for 24 hours. Whole cell lysates were collected for Western blot assays. Histone H2A was used as a loading control. The activation and inactivation folds indicated below each lane were normalized against those in mock controls (cell alone). The levels of indicated proteins in the mock control were considered 1-fold. (B) Representative images of vero cells transfected with plasmids overexpressing EGFP-Tpr. Vero cells were transfected with negative controls (mock and vector only) or EGFP-Tpr plasmid for 48 hours and then visualized by immunofluorescence following nuclear DAPI staining. In addition, another set of cells were transfected with EGFP-Tpr for 48 hours and then transfected with p17 for 24 hours. Cells were fixed and processed for immunofluorescence staining of p17. Colocalization of EGFP-Tpr and p17 was observed under a fluorescence microscope. Scale bar: 10 um. (C) To examine whether AKT kinase activity is directly required for p21, we used an Akt DN to inhibit AKT in Vero cells. The levels of p-p21 (T145) in the nucleus were examined in ARV-infected and p17-transfected cells in the presence or absence of an Akt DN. Histone H2A was used as a loading control.
Mentions: Importantly, the elevation in p-53 (S15), p-p21 (T145), and PTEN levels in the nucleus were also seen not only in Tpr-depleted cells but also in ARV-infected and p17-transfected cells (Fig 3D; S1D Fig). Furthermore, the effects of both p17-transfection and ARV-infection could be abolished by overexpression of EGFP-Tpr (Fig 4A), suggesting that p17 causes the increase in p-53 (S15), p-p21 (T145), and PTEN levels by a downregulation of Tpr. In the present study, representative images of Vero cells transfected with plasmids overexpressing EGFP-Tpr was shown in Fig 4B. Colocalization of EGFP-Tpr and p17 was observed under a fluorescence microscope (Fig 4B). Since both p21 and PTEN are the downstream targets of p53 [44, 45], upregulation of PTEN and p21 in Tpr-depleted cells was due to activation of p53. Our observations were consistent with a previous report suggesting that depletion of Tpr reduced nuclear pore formation and nucleo-cytoplasmic trafficking activities, thereby causing p53 nuclear accumulation, which induced specific downstream target genes of the p53 pathway [9, 46].

Bottom Line: To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner.The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4.Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan.

ABSTRACT
Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

No MeSH data available.


Related in: MedlinePlus