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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 specifically interacts with Tpr and blocks p53 binding to Tpr.(A) To study whether p17 and p17 mutants can downregulate Tpr expression, the levels of Tpr, p-p53, p-p21 were examined in mock-infected and ARV-infected cells as well as in pcDNA3.1-p17-, pcDNA3.1- (vector only), and p17 mutants- transfected cells. Tricine-SDS-PAGE for separating proteins in the low mass range was used. After transfection of the p17 gene and the truncated version of p17 into vero cells for 24 hours, all these proteins were separated by Tricine-SDS-PAGE, followed by Western blot assay with the anti-Flag antibody. (B) Upper panel: to examine whether p17 and p17 mutants affect the binding of p53 to Tpr, the amount of p53 and Tpr association in mock (Vero cells only), ARV-infection, pcDNA3.1-p17- and p17 mutants-transfection were examined. Lower panel: To carry out a rescue assay, cells were transfected with the indicated plasmids or infected with ARV for 24 hours. About 500 ug of cellular proteins was incubated with 4 ug of anti-p53 antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blotting, and then proteins were detected with Tpr or p53 antibody, respectively. The protein levels were normalized to those for β-actin. Results were obtained from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at mock controls. The levels of indicated proteins at the mock control were considered 1-fold.
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pone.0133699.g005: p17 specifically interacts with Tpr and blocks p53 binding to Tpr.(A) To study whether p17 and p17 mutants can downregulate Tpr expression, the levels of Tpr, p-p53, p-p21 were examined in mock-infected and ARV-infected cells as well as in pcDNA3.1-p17-, pcDNA3.1- (vector only), and p17 mutants- transfected cells. Tricine-SDS-PAGE for separating proteins in the low mass range was used. After transfection of the p17 gene and the truncated version of p17 into vero cells for 24 hours, all these proteins were separated by Tricine-SDS-PAGE, followed by Western blot assay with the anti-Flag antibody. (B) Upper panel: to examine whether p17 and p17 mutants affect the binding of p53 to Tpr, the amount of p53 and Tpr association in mock (Vero cells only), ARV-infection, pcDNA3.1-p17- and p17 mutants-transfection were examined. Lower panel: To carry out a rescue assay, cells were transfected with the indicated plasmids or infected with ARV for 24 hours. About 500 ug of cellular proteins was incubated with 4 ug of anti-p53 antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blotting, and then proteins were detected with Tpr or p53 antibody, respectively. The protein levels were normalized to those for β-actin. Results were obtained from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at mock controls. The levels of indicated proteins at the mock control were considered 1-fold.

Mentions: Many studies have suggested that Tpr plays a role in nuclear protein export [3–5] and is one of the nucleoporin proteins regulating p53 nuclear-cytoplasmic trafficking [9]. Consistent with recent reports, p17-mediated downregulation of Tpr affects p53 nucleocytoplasmic trafficking, leading to p53 nuclear accumulation and activation. In this work, Tricine-SDS-PAGE for separating proteins in the low mass range was used [40]. After transfection of the Flag-tagged p17 gene and all truncated versions of p17 into vero cells for 24 hours, all these proteins were separated by Tricine-SDS-PAGE, followed by Western blot assay with the anti-Flag antibody (Fig 5A, lanes 4–7). Predictably, undetectable proteins were observed in vector only, mock-infected, and ARV-infected cells. The expression of p17 in ARV-infected cells was detected when the anti-p17 antibody was used (data not shown). As shown in Fig 5A (lanes 5–7), all truncated versions of p17 (1–118, 27–146, and 119–146) only slightly reduced the expression level of Tpr compared to negative controls and did not cause accumulation of p-p53 and p- p21 in the nucleus compared to both ARV infection and p17 transfection. This finding suggests that p17 leads to nuclear accumulation of p53 and p21 by a mechanism of transcriptional downregulation of Tpr. As the reciprocal co-immunoprecipitation experiments detailed above could not rule out the possibility that the reduced amounts of Tpr-p17 binding was due to the decrease in Tpr level, therefore, we examined the amount of binding of p53 to Tpr in cells with different treatments. In p53 co-immunoprecipitated complexes, the amount of p53-Tpr binding was dramatically reduced in ARV-infected, pcDNA3.1-p17-, and p17 mutants-transfected Vero cells relative to mock controls (cell alone and vector only) (Fig 5B, upper panel, lanes 3–4). Interestingly, the truncated versions of p17 (27–146 and 119–146) significantly diminished the amount of p53-Tpr binding (Fig 5B, upper panel, lanes 5, 7) while the amount of Tpr-p53 binding was not altered in p17 (1–118) transfection which lacks the NLS in our co-immunoprecipitation assays. Since p17 mutants (27–146 and 119–146) did not reduce the expression level of Tpr, therefore, these p17 mutants block p53 binding to Tpr by competing with p53. To further validate our findings, we employed a rescue assay by overexpressing EGFP-Tpr in both ARV-infected and p17-transfected vero cells. The effects of both ARV-infection and p17-transfection could be abolished by overexpression of EGFP-Tpr (Fig 5B, lower panel, lanes 5–6), suggesting that p17 downregulates Tpr expression level reducing the amounts of p53-Tpr binding. Taken together, we conclude that p17 functions as a Tpr suppressor by at least two independent mechanisms, which include p17-Tpr interaction and transcriptional downregulation of Tpr, thereby blocking p53 nuclear export and causing p53 nuclear accumulation.


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 specifically interacts with Tpr and blocks p53 binding to Tpr.(A) To study whether p17 and p17 mutants can downregulate Tpr expression, the levels of Tpr, p-p53, p-p21 were examined in mock-infected and ARV-infected cells as well as in pcDNA3.1-p17-, pcDNA3.1- (vector only), and p17 mutants- transfected cells. Tricine-SDS-PAGE for separating proteins in the low mass range was used. After transfection of the p17 gene and the truncated version of p17 into vero cells for 24 hours, all these proteins were separated by Tricine-SDS-PAGE, followed by Western blot assay with the anti-Flag antibody. (B) Upper panel: to examine whether p17 and p17 mutants affect the binding of p53 to Tpr, the amount of p53 and Tpr association in mock (Vero cells only), ARV-infection, pcDNA3.1-p17- and p17 mutants-transfection were examined. Lower panel: To carry out a rescue assay, cells were transfected with the indicated plasmids or infected with ARV for 24 hours. About 500 ug of cellular proteins was incubated with 4 ug of anti-p53 antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blotting, and then proteins were detected with Tpr or p53 antibody, respectively. The protein levels were normalized to those for β-actin. Results were obtained from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at mock controls. The levels of indicated proteins at the mock control were considered 1-fold.
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Related In: Results  -  Collection

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pone.0133699.g005: p17 specifically interacts with Tpr and blocks p53 binding to Tpr.(A) To study whether p17 and p17 mutants can downregulate Tpr expression, the levels of Tpr, p-p53, p-p21 were examined in mock-infected and ARV-infected cells as well as in pcDNA3.1-p17-, pcDNA3.1- (vector only), and p17 mutants- transfected cells. Tricine-SDS-PAGE for separating proteins in the low mass range was used. After transfection of the p17 gene and the truncated version of p17 into vero cells for 24 hours, all these proteins were separated by Tricine-SDS-PAGE, followed by Western blot assay with the anti-Flag antibody. (B) Upper panel: to examine whether p17 and p17 mutants affect the binding of p53 to Tpr, the amount of p53 and Tpr association in mock (Vero cells only), ARV-infection, pcDNA3.1-p17- and p17 mutants-transfection were examined. Lower panel: To carry out a rescue assay, cells were transfected with the indicated plasmids or infected with ARV for 24 hours. About 500 ug of cellular proteins was incubated with 4 ug of anti-p53 antibody at 4°C overnight. The immunoprecipitated proteins were separated by SDS-PAGE followed by Western blotting, and then proteins were detected with Tpr or p53 antibody, respectively. The protein levels were normalized to those for β-actin. Results were obtained from three independent experiments. The activation and inactivation folds indicated below each lane were normalized against those at mock controls. The levels of indicated proteins at the mock control were considered 1-fold.
Mentions: Many studies have suggested that Tpr plays a role in nuclear protein export [3–5] and is one of the nucleoporin proteins regulating p53 nuclear-cytoplasmic trafficking [9]. Consistent with recent reports, p17-mediated downregulation of Tpr affects p53 nucleocytoplasmic trafficking, leading to p53 nuclear accumulation and activation. In this work, Tricine-SDS-PAGE for separating proteins in the low mass range was used [40]. After transfection of the Flag-tagged p17 gene and all truncated versions of p17 into vero cells for 24 hours, all these proteins were separated by Tricine-SDS-PAGE, followed by Western blot assay with the anti-Flag antibody (Fig 5A, lanes 4–7). Predictably, undetectable proteins were observed in vector only, mock-infected, and ARV-infected cells. The expression of p17 in ARV-infected cells was detected when the anti-p17 antibody was used (data not shown). As shown in Fig 5A (lanes 5–7), all truncated versions of p17 (1–118, 27–146, and 119–146) only slightly reduced the expression level of Tpr compared to negative controls and did not cause accumulation of p-p53 and p- p21 in the nucleus compared to both ARV infection and p17 transfection. This finding suggests that p17 leads to nuclear accumulation of p53 and p21 by a mechanism of transcriptional downregulation of Tpr. As the reciprocal co-immunoprecipitation experiments detailed above could not rule out the possibility that the reduced amounts of Tpr-p17 binding was due to the decrease in Tpr level, therefore, we examined the amount of binding of p53 to Tpr in cells with different treatments. In p53 co-immunoprecipitated complexes, the amount of p53-Tpr binding was dramatically reduced in ARV-infected, pcDNA3.1-p17-, and p17 mutants-transfected Vero cells relative to mock controls (cell alone and vector only) (Fig 5B, upper panel, lanes 3–4). Interestingly, the truncated versions of p17 (27–146 and 119–146) significantly diminished the amount of p53-Tpr binding (Fig 5B, upper panel, lanes 5, 7) while the amount of Tpr-p53 binding was not altered in p17 (1–118) transfection which lacks the NLS in our co-immunoprecipitation assays. Since p17 mutants (27–146 and 119–146) did not reduce the expression level of Tpr, therefore, these p17 mutants block p53 binding to Tpr by competing with p53. To further validate our findings, we employed a rescue assay by overexpressing EGFP-Tpr in both ARV-infected and p17-transfected vero cells. The effects of both ARV-infection and p17-transfection could be abolished by overexpression of EGFP-Tpr (Fig 5B, lower panel, lanes 5–6), suggesting that p17 downregulates Tpr expression level reducing the amounts of p53-Tpr binding. Taken together, we conclude that p17 functions as a Tpr suppressor by at least two independent mechanisms, which include p17-Tpr interaction and transcriptional downregulation of Tpr, thereby blocking p53 nuclear export and causing p53 nuclear accumulation.

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