Limits...
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 stabilizes PTEN by promoting Rak-PTEN association and by stimulating phosphorylation of PTEN to protect PTEN from NEDD4-1 targeting.(A) In reciprocal co-immunoprecipation experiments, the amounts of Rak and PTEN association were examined in ARV-infected or p17-transfected cells. Western blot assay of PTEN, and NEDD4-1 contained in PTEN or Rak immunoprecipates was carried out. The p17-transfected, mock-transfected, mock-infected, and ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection for Western blot assays. Data shown represent the mean± SD calculated from three independent experiments. The amounts of Rak-PTEN and NEDD4-1-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (B) In reciprocal co-immunoprecipation experiments, the binding of β-arrestin to PTEN was examined in either ARV-infected or p17-transfected Vero cells. Western blot of β-arrestin, PTEN, and NEDD4-1 contained in PTEN or β-arrestin immunoprecipates was carried out. Data shown represent the mean± SD calculated from three independent experiments. The amounts of β-arrestin-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (C) In coimmunoprecipation experiments, the binding of β-arrestin to PTEN was examined in p17-transfected Vero cells. Western blot of PTEN and β-arrestin contained in PTEN immunoprecipates was carried out in presence and absence of Rock-1 shRNA. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at vector only. The level of indicated proteins at vector only was considered 1-fold. (D) The interaction between PTEN and β-arrestin as well as between NEDD4-1 and PTEN were examined in p17-transfected and mock-transfected Vero cells. Proteins immunoprecipated with an anti-PTEN antibody from Vero cell lysates treated with Rak shRNA, Y-27632, and TBB, respectively were resolved by SDS-PAGE and immunbloted with the indicated antibodies. Data shown represent the mean± SD calculated from three independent experiments. (E) Cells were co-transfected with pcDNA3.1-p17 plasmid with either Rak or Rock-1 shRNA for 24 hours. The cells were harvested and washed twice in PBS buffer and scraped in 200 μl of lysis buffer. About 500 ug of cellular proteins was incubated with 4 ug of anti-PTEN antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot assay, and then proteins were detected with anti-ubiquitin antibody. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. (F) A model illustrates the PTEN regulation by p17.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526660&req=5

pone.0133699.g007: p17 stabilizes PTEN by promoting Rak-PTEN association and by stimulating phosphorylation of PTEN to protect PTEN from NEDD4-1 targeting.(A) In reciprocal co-immunoprecipation experiments, the amounts of Rak and PTEN association were examined in ARV-infected or p17-transfected cells. Western blot assay of PTEN, and NEDD4-1 contained in PTEN or Rak immunoprecipates was carried out. The p17-transfected, mock-transfected, mock-infected, and ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection for Western blot assays. Data shown represent the mean± SD calculated from three independent experiments. The amounts of Rak-PTEN and NEDD4-1-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (B) In reciprocal co-immunoprecipation experiments, the binding of β-arrestin to PTEN was examined in either ARV-infected or p17-transfected Vero cells. Western blot of β-arrestin, PTEN, and NEDD4-1 contained in PTEN or β-arrestin immunoprecipates was carried out. Data shown represent the mean± SD calculated from three independent experiments. The amounts of β-arrestin-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (C) In coimmunoprecipation experiments, the binding of β-arrestin to PTEN was examined in p17-transfected Vero cells. Western blot of PTEN and β-arrestin contained in PTEN immunoprecipates was carried out in presence and absence of Rock-1 shRNA. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at vector only. The level of indicated proteins at vector only was considered 1-fold. (D) The interaction between PTEN and β-arrestin as well as between NEDD4-1 and PTEN were examined in p17-transfected and mock-transfected Vero cells. Proteins immunoprecipated with an anti-PTEN antibody from Vero cell lysates treated with Rak shRNA, Y-27632, and TBB, respectively were resolved by SDS-PAGE and immunbloted with the indicated antibodies. Data shown represent the mean± SD calculated from three independent experiments. (E) Cells were co-transfected with pcDNA3.1-p17 plasmid with either Rak or Rock-1 shRNA for 24 hours. The cells were harvested and washed twice in PBS buffer and scraped in 200 μl of lysis buffer. About 500 ug of cellular proteins was incubated with 4 ug of anti-PTEN antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot assay, and then proteins were detected with anti-ubiquitin antibody. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. (F) A model illustrates the PTEN regulation by p17.

Mentions: A previous study on PTEN protein stability has demonstrated that it is regulated by ubiquitin-mediated proteasome degradation through the E3 ligase NEDD4-1 [53]. Phosphorylation of PTEN by either CK2 or Rak was reported to enhance PTEN stability [48–50]. To test whether p17 stabilizes PTEN by enhancing Rak binding to PTEN, reciprocal coimmunoprecipation assays were carried out. Our results revealed that the increase in the interaction between Rak and PTEN was seen in ARV-infected and p17-transfected cells (up to 3.8±0.15 and 4.5±0.2 folds, respectively) (Fig 7A), thereby inhibiting NEDD4-1 targeting to PTEN (down to 2.8±0.3 and 3.1± 0.25 folds, respectively) (Fig 7A). Furtherore, an increase in β-arrestin-PTEN association was also observed (up to 3.5±0.25 and 6±0.2 folds, respectively) (Fig 7B), accompanied by a decreased amount of NEDD4-1-PTEN association.


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 stabilizes PTEN by promoting Rak-PTEN association and by stimulating phosphorylation of PTEN to protect PTEN from NEDD4-1 targeting.(A) In reciprocal co-immunoprecipation experiments, the amounts of Rak and PTEN association were examined in ARV-infected or p17-transfected cells. Western blot assay of PTEN, and NEDD4-1 contained in PTEN or Rak immunoprecipates was carried out. The p17-transfected, mock-transfected, mock-infected, and ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection for Western blot assays. Data shown represent the mean± SD calculated from three independent experiments. The amounts of Rak-PTEN and NEDD4-1-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (B) In reciprocal co-immunoprecipation experiments, the binding of β-arrestin to PTEN was examined in either ARV-infected or p17-transfected Vero cells. Western blot of β-arrestin, PTEN, and NEDD4-1 contained in PTEN or β-arrestin immunoprecipates was carried out. Data shown represent the mean± SD calculated from three independent experiments. The amounts of β-arrestin-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (C) In coimmunoprecipation experiments, the binding of β-arrestin to PTEN was examined in p17-transfected Vero cells. Western blot of PTEN and β-arrestin contained in PTEN immunoprecipates was carried out in presence and absence of Rock-1 shRNA. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at vector only. The level of indicated proteins at vector only was considered 1-fold. (D) The interaction between PTEN and β-arrestin as well as between NEDD4-1 and PTEN were examined in p17-transfected and mock-transfected Vero cells. Proteins immunoprecipated with an anti-PTEN antibody from Vero cell lysates treated with Rak shRNA, Y-27632, and TBB, respectively were resolved by SDS-PAGE and immunbloted with the indicated antibodies. Data shown represent the mean± SD calculated from three independent experiments. (E) Cells were co-transfected with pcDNA3.1-p17 plasmid with either Rak or Rock-1 shRNA for 24 hours. The cells were harvested and washed twice in PBS buffer and scraped in 200 μl of lysis buffer. About 500 ug of cellular proteins was incubated with 4 ug of anti-PTEN antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot assay, and then proteins were detected with anti-ubiquitin antibody. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. (F) A model illustrates the PTEN regulation by p17.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526660&req=5

pone.0133699.g007: p17 stabilizes PTEN by promoting Rak-PTEN association and by stimulating phosphorylation of PTEN to protect PTEN from NEDD4-1 targeting.(A) In reciprocal co-immunoprecipation experiments, the amounts of Rak and PTEN association were examined in ARV-infected or p17-transfected cells. Western blot assay of PTEN, and NEDD4-1 contained in PTEN or Rak immunoprecipates was carried out. The p17-transfected, mock-transfected, mock-infected, and ARV-infected cells were collected at either 24 hpi or 24 hours post-transfection for Western blot assays. Data shown represent the mean± SD calculated from three independent experiments. The amounts of Rak-PTEN and NEDD4-1-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (B) In reciprocal co-immunoprecipation experiments, the binding of β-arrestin to PTEN was examined in either ARV-infected or p17-transfected Vero cells. Western blot of β-arrestin, PTEN, and NEDD4-1 contained in PTEN or β-arrestin immunoprecipates was carried out. Data shown represent the mean± SD calculated from three independent experiments. The amounts of β-arrestin-PTEN associations were normalized against those at mock-transfection and mock-infection. The level of mock controls was considered 1-fold. (C) In coimmunoprecipation experiments, the binding of β-arrestin to PTEN was examined in p17-transfected Vero cells. Western blot of PTEN and β-arrestin contained in PTEN immunoprecipates was carried out in presence and absence of Rock-1 shRNA. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at vector only. The level of indicated proteins at vector only was considered 1-fold. (D) The interaction between PTEN and β-arrestin as well as between NEDD4-1 and PTEN were examined in p17-transfected and mock-transfected Vero cells. Proteins immunoprecipated with an anti-PTEN antibody from Vero cell lysates treated with Rak shRNA, Y-27632, and TBB, respectively were resolved by SDS-PAGE and immunbloted with the indicated antibodies. Data shown represent the mean± SD calculated from three independent experiments. (E) Cells were co-transfected with pcDNA3.1-p17 plasmid with either Rak or Rock-1 shRNA for 24 hours. The cells were harvested and washed twice in PBS buffer and scraped in 200 μl of lysis buffer. About 500 ug of cellular proteins was incubated with 4 ug of anti-PTEN antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blot assay, and then proteins were detected with anti-ubiquitin antibody. Rabbit IgG was used as a negative control. The representative data are from three independent experiments. (F) A model illustrates the PTEN regulation by p17.
Mentions: A previous study on PTEN protein stability has demonstrated that it is regulated by ubiquitin-mediated proteasome degradation through the E3 ligase NEDD4-1 [53]. Phosphorylation of PTEN by either CK2 or Rak was reported to enhance PTEN stability [48–50]. To test whether p17 stabilizes PTEN by enhancing Rak binding to PTEN, reciprocal coimmunoprecipation assays were carried out. Our results revealed that the increase in the interaction between Rak and PTEN was seen in ARV-infected and p17-transfected cells (up to 3.8±0.15 and 4.5±0.2 folds, respectively) (Fig 7A), thereby inhibiting NEDD4-1 targeting to PTEN (down to 2.8±0.3 and 3.1± 0.25 folds, respectively) (Fig 7A). Furtherore, an increase in β-arrestin-PTEN association was also observed (up to 3.5±0.25 and 6±0.2 folds, respectively) (Fig 7B), accompanied by a decreased amount of NEDD4-1-PTEN association.

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