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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

p17 positively regulates PTEN and Rak expression and drives PTEN translocation from cytoplasm into plasma membrane.(A) The p17, p53, PTEN, Rak, and GADPH mRNA levels were examined by semi-quantitative RT-PCR in ARV-infected and p17 transfected Vero cells in the presence or absence of indicated p53 shRNA. In semi-quantitative RT-PCR amplification of the p17, p53, PTEN, Rak, and GADPH genes, Vero cells were transfected with p17 or infected with ARV at an MOI of 10. The p17-transfected or ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection, and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock control (cell alone) was included as a negative control. Graph shown represents the mean± SD calculated from three independent experiments. (B) The p17, p53, p-PTEN, cytoplasmic PTEN, membrane-associated PTEN, Rak, and NEDD4-1 levels were examined in p17-transfected Vero cells and mock control cells. (C) To confirm p53 regulated PTEN in Vero and DF-1 cells, the p-p53, p53, and PTEN levels were examined in pcDNA3.1-p17 and p53 shRNA-cotransfected cells. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17, p53 shRNA, scramble shRNA, and pGFP-V-RS (vector only) for 24 hours (D) Effects of Y-27632 and TBB inhibitors on p17-mediated PTEN and β-arrestin translocation. In the presence and absence of indicated Y-27632 and TBB, the expression levels of p17, p-PTEN, cytoplasmic PTEN, membrane-associated PTEN, cytoplasmic β-arrestin, membrane-associated β-arrestin, and Rock-1 were examined in p17-transfected Vero cells and negative control (cell alone). Vero cells were pretreated with either Y-27632 (10 μM) or TBB (5 μM) for 1 hour followed by transfection with pcDNA3.1-p1 for 24 hours at 37°C. Graph on right panel shows the relative level of PTEN and β-arrestin in membrane in p17-transfected cells in the presence of Y-27632 or TBB versus cell alone. Results were obtained from three independent experiment, error bars indicate the means± SD. The protein levels were normalized to those for β-actin or Na+/K+ ATPase. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panels B and D) or mock control (panel C). The levels of indicated proteins at 0 h or in mock transfection (vector only) were considered 1-fold. The representative data are from three independent experiments.
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pone.0133699.g006: p17 positively regulates PTEN and Rak expression and drives PTEN translocation from cytoplasm into plasma membrane.(A) The p17, p53, PTEN, Rak, and GADPH mRNA levels were examined by semi-quantitative RT-PCR in ARV-infected and p17 transfected Vero cells in the presence or absence of indicated p53 shRNA. In semi-quantitative RT-PCR amplification of the p17, p53, PTEN, Rak, and GADPH genes, Vero cells were transfected with p17 or infected with ARV at an MOI of 10. The p17-transfected or ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection, and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock control (cell alone) was included as a negative control. Graph shown represents the mean± SD calculated from three independent experiments. (B) The p17, p53, p-PTEN, cytoplasmic PTEN, membrane-associated PTEN, Rak, and NEDD4-1 levels were examined in p17-transfected Vero cells and mock control cells. (C) To confirm p53 regulated PTEN in Vero and DF-1 cells, the p-p53, p53, and PTEN levels were examined in pcDNA3.1-p17 and p53 shRNA-cotransfected cells. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17, p53 shRNA, scramble shRNA, and pGFP-V-RS (vector only) for 24 hours (D) Effects of Y-27632 and TBB inhibitors on p17-mediated PTEN and β-arrestin translocation. In the presence and absence of indicated Y-27632 and TBB, the expression levels of p17, p-PTEN, cytoplasmic PTEN, membrane-associated PTEN, cytoplasmic β-arrestin, membrane-associated β-arrestin, and Rock-1 were examined in p17-transfected Vero cells and negative control (cell alone). Vero cells were pretreated with either Y-27632 (10 μM) or TBB (5 μM) for 1 hour followed by transfection with pcDNA3.1-p1 for 24 hours at 37°C. Graph on right panel shows the relative level of PTEN and β-arrestin in membrane in p17-transfected cells in the presence of Y-27632 or TBB versus cell alone. Results were obtained from three independent experiment, error bars indicate the means± SD. The protein levels were normalized to those for β-actin or Na+/K+ ATPase. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panels B and D) or mock control (panel C). The levels of indicated proteins at 0 h or in mock transfection (vector only) were considered 1-fold. The representative data are from three independent experiments.

Mentions: Having demonstrated that p17 leads to p53 accumulation and activation in the nucleus, we next intended to examine if the mRNA level of p53 and its downstream molecule PTEN were upregulated by p17. In the present study, PTEN transcripts were significantly elevated in ARV-infected and p17-transfected Vero cells (up to 2.45± 0.08 and 2.4 ± 0.1 folds, respectively) (Fig 6A). It is clear that PTEN is one of the p53 downstream targets. Its transcription induction by p17 is very likely through activation of p53. No significant change of p53 transcripts was observed in ARV-infected and p17-transfected Vero cells, indicating that p17 did not affect p53 gene transcription (Fig 6A). Importantly, the p-p53 levels were elevated in both ARV-infected and pcDNA3.1-p17 transfected cells (Figs 3A and 6B; S1A Fig). Furthermore, we also found that Rak transcripts were elevated in both ARV infected and p17-transfected Vero cells (up to 2.3 ±0.1 and 2.25 ±0.05 folds, respectively) (Fig 6A). In this study, only PTEN transcripts were diminished in ARV-infected Vero cells in the presence of p53 shRNA (Fig 6A), indicating that PTEN, but not Rak, was upregulated by p53. p53 Knockdown reduced the PTEN levels, further confirming that p53 positively regulates the PTEN level in our studies (Fig 6C).


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)

p17 positively regulates PTEN and Rak expression and drives PTEN translocation from cytoplasm into plasma membrane.(A) The p17, p53, PTEN, Rak, and GADPH mRNA levels were examined by semi-quantitative RT-PCR in ARV-infected and p17 transfected Vero cells in the presence or absence of indicated p53 shRNA. In semi-quantitative RT-PCR amplification of the p17, p53, PTEN, Rak, and GADPH genes, Vero cells were transfected with p17 or infected with ARV at an MOI of 10. The p17-transfected or ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection, and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock control (cell alone) was included as a negative control. Graph shown represents the mean± SD calculated from three independent experiments. (B) The p17, p53, p-PTEN, cytoplasmic PTEN, membrane-associated PTEN, Rak, and NEDD4-1 levels were examined in p17-transfected Vero cells and mock control cells. (C) To confirm p53 regulated PTEN in Vero and DF-1 cells, the p-p53, p53, and PTEN levels were examined in pcDNA3.1-p17 and p53 shRNA-cotransfected cells. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17, p53 shRNA, scramble shRNA, and pGFP-V-RS (vector only) for 24 hours (D) Effects of Y-27632 and TBB inhibitors on p17-mediated PTEN and β-arrestin translocation. In the presence and absence of indicated Y-27632 and TBB, the expression levels of p17, p-PTEN, cytoplasmic PTEN, membrane-associated PTEN, cytoplasmic β-arrestin, membrane-associated β-arrestin, and Rock-1 were examined in p17-transfected Vero cells and negative control (cell alone). Vero cells were pretreated with either Y-27632 (10 μM) or TBB (5 μM) for 1 hour followed by transfection with pcDNA3.1-p1 for 24 hours at 37°C. Graph on right panel shows the relative level of PTEN and β-arrestin in membrane in p17-transfected cells in the presence of Y-27632 or TBB versus cell alone. Results were obtained from three independent experiment, error bars indicate the means± SD. The protein levels were normalized to those for β-actin or Na+/K+ ATPase. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panels B and D) or mock control (panel C). The levels of indicated proteins at 0 h or in mock transfection (vector only) were considered 1-fold. The representative data are from three independent experiments.
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Related In: Results  -  Collection

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pone.0133699.g006: p17 positively regulates PTEN and Rak expression and drives PTEN translocation from cytoplasm into plasma membrane.(A) The p17, p53, PTEN, Rak, and GADPH mRNA levels were examined by semi-quantitative RT-PCR in ARV-infected and p17 transfected Vero cells in the presence or absence of indicated p53 shRNA. In semi-quantitative RT-PCR amplification of the p17, p53, PTEN, Rak, and GADPH genes, Vero cells were transfected with p17 or infected with ARV at an MOI of 10. The p17-transfected or ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection, and total RNAs were extracted for semi-quantitative RT-PCR. After electrophoretic separation in an agarose gel, PCR products were stained with ethidium bromide. Mock control (cell alone) was included as a negative control. Graph shown represents the mean± SD calculated from three independent experiments. (B) The p17, p53, p-PTEN, cytoplasmic PTEN, membrane-associated PTEN, Rak, and NEDD4-1 levels were examined in p17-transfected Vero cells and mock control cells. (C) To confirm p53 regulated PTEN in Vero and DF-1 cells, the p-p53, p53, and PTEN levels were examined in pcDNA3.1-p17 and p53 shRNA-cotransfected cells. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17, p53 shRNA, scramble shRNA, and pGFP-V-RS (vector only) for 24 hours (D) Effects of Y-27632 and TBB inhibitors on p17-mediated PTEN and β-arrestin translocation. In the presence and absence of indicated Y-27632 and TBB, the expression levels of p17, p-PTEN, cytoplasmic PTEN, membrane-associated PTEN, cytoplasmic β-arrestin, membrane-associated β-arrestin, and Rock-1 were examined in p17-transfected Vero cells and negative control (cell alone). Vero cells were pretreated with either Y-27632 (10 μM) or TBB (5 μM) for 1 hour followed by transfection with pcDNA3.1-p1 for 24 hours at 37°C. Graph on right panel shows the relative level of PTEN and β-arrestin in membrane in p17-transfected cells in the presence of Y-27632 or TBB versus cell alone. Results were obtained from three independent experiment, error bars indicate the means± SD. The protein levels were normalized to those for β-actin or Na+/K+ ATPase. The activation and inactivation folds indicated below each lane were normalized against those at 0 h (panels B and D) or mock control (panel C). The levels of indicated proteins at 0 h or in mock transfection (vector only) were considered 1-fold. The representative data are from three independent experiments.
Mentions: Having demonstrated that p17 leads to p53 accumulation and activation in the nucleus, we next intended to examine if the mRNA level of p53 and its downstream molecule PTEN were upregulated by p17. In the present study, PTEN transcripts were significantly elevated in ARV-infected and p17-transfected Vero cells (up to 2.45± 0.08 and 2.4 ± 0.1 folds, respectively) (Fig 6A). It is clear that PTEN is one of the p53 downstream targets. Its transcription induction by p17 is very likely through activation of p53. No significant change of p53 transcripts was observed in ARV-infected and p17-transfected Vero cells, indicating that p17 did not affect p53 gene transcription (Fig 6A). Importantly, the p-p53 levels were elevated in both ARV-infected and pcDNA3.1-p17 transfected cells (Figs 3A and 6B; S1A Fig). Furthermore, we also found that Rak transcripts were elevated in both ARV infected and p17-transfected Vero cells (up to 2.3 ±0.1 and 2.25 ±0.05 folds, respectively) (Fig 6A). In this study, only PTEN transcripts were diminished in ARV-infected Vero cells in the presence of p53 shRNA (Fig 6A), indicating that PTEN, but not Rak, was upregulated by p53. p53 Knockdown reduced the PTEN levels, further confirming that p53 positively regulates the PTEN level in our studies (Fig 6C).

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