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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 negatively regulates ERK and cyclin D1 and positively regulates Rb through Tpr/p53/PTEN and Tpr/p53/p21 pathways.(A-B)Vero cells were transfected with pcDNA3.1-p17 or pcDNA3.1 (vector only) for 24 hours. The results of Western blot analysis of cellular fractions from the cytoplasm and nucleus (A) or whole cell lysates (B) at the indicated time points are shown. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. (C) To study whether the p17 mutant (1–118) which does not possess a NLS can affect the levels of p-ERK, cyclin D1, and CDK4 in the nucleus, vero cells were transfected with the negative control p17 mutant (1–118) for 24 hours. (D) To confirm whether PTEN is the upstream signaling that regulates the above molecules, shRNA-mediated blockade of PTEN was performed. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17 and PTEN shRNAs for 24 hours followed by Western blot analysis with indicated antibodies. In the negative controls, cells were also co-transfected with p17 and respective negative controls (pGFP-V-RS and Scramble shRNA plasmids) for 24 hours. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. The protein levels were normalized to that for β-actin (panels A, B, C) or Histone H2A (panel A). The activation and inactivation folds indicated below each lane were normalized against those at 0 h or in mock control. The levels of indicated proteins at 0 h or in mock control (cell only) were considered 1-fold. The representative data are from three independent experiments.
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pone.0133699.g008: p17 negatively regulates ERK and cyclin D1 and positively regulates Rb through Tpr/p53/PTEN and Tpr/p53/p21 pathways.(A-B)Vero cells were transfected with pcDNA3.1-p17 or pcDNA3.1 (vector only) for 24 hours. The results of Western blot analysis of cellular fractions from the cytoplasm and nucleus (A) or whole cell lysates (B) at the indicated time points are shown. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. (C) To study whether the p17 mutant (1–118) which does not possess a NLS can affect the levels of p-ERK, cyclin D1, and CDK4 in the nucleus, vero cells were transfected with the negative control p17 mutant (1–118) for 24 hours. (D) To confirm whether PTEN is the upstream signaling that regulates the above molecules, shRNA-mediated blockade of PTEN was performed. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17 and PTEN shRNAs for 24 hours followed by Western blot analysis with indicated antibodies. In the negative controls, cells were also co-transfected with p17 and respective negative controls (pGFP-V-RS and Scramble shRNA plasmids) for 24 hours. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. The protein levels were normalized to that for β-actin (panels A, B, C) or Histone H2A (panel A). The activation and inactivation folds indicated below each lane were normalized against those at 0 h or in mock control. The levels of indicated proteins at 0 h or in mock control (cell only) were considered 1-fold. The representative data are from three independent experiments.

Mentions: As seen from the the previous data showing that p17 activated PTEN, we next wanted to investigate whether its downstream molecules were suppressed. Nuclear PTEN is essential for tumor suppression and its nuclear import is mediated by its monoubiquitination [54]. In this study, the elevation in nuclear PTEN was found in p17-expressing cells. Consistent with a previous study [55], the increased levels of PTEN were correlated with a decrease in p-ERK and cyclin D1 levels in the nucleus in a time-dependent manner, as revealed by Western blot (Fig 8A; S3A Fig). Based on current knowledge, control of the G1/S phases of cell cycle transition is largely a matter of regulating a set of specific CDK activities. In mammalian cells, the G1/S specific CDK activities are composed of complexes between D type cyclins and either CDK4 or CDK6. A previous study has suggested that cyclin D1-CDK4 phosphorylates Rb in vivo [56]. Kitagawa and co-workers suggested that the S780 in Rb is phosphorylated during the G1 phase in a cell cycle-dependent manner and that Rb phosphorylated at S780 cannot bind to E2F-1 in vivo [56]. Importantly, this study further demonstrates that p17 negatively regulates Tpr leading to positive regulation of p53, PTEN, and p21 with the concomitant decrease in p-ERK, cyclin D1, and CDK4 levels in both Vero and DF-1 cells in a time-dependent manner, thereby resulting in Rb (S780) dephosphorylation/activation (Fig 8A and 8B; S3A and S3B Figs). The level of E2F-1 was not altered. In addition, a dramatic reduction in the cyclin D1 level in the cytoplasm and the nucleus was seen in p17-transfected cells (Fig 8A; S3A Fig). The levels of p-ERK, cyclin D1, CDK 4, and p-Rb were reversed in p17 (1–118)-transfected cells (Fig 8C) as compared to p17 transfection. The levels of PTEN and its downstream molecules are summarized in S1 Table.


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 negatively regulates ERK and cyclin D1 and positively regulates Rb through Tpr/p53/PTEN and Tpr/p53/p21 pathways.(A-B)Vero cells were transfected with pcDNA3.1-p17 or pcDNA3.1 (vector only) for 24 hours. The results of Western blot analysis of cellular fractions from the cytoplasm and nucleus (A) or whole cell lysates (B) at the indicated time points are shown. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. (C) To study whether the p17 mutant (1–118) which does not possess a NLS can affect the levels of p-ERK, cyclin D1, and CDK4 in the nucleus, vero cells were transfected with the negative control p17 mutant (1–118) for 24 hours. (D) To confirm whether PTEN is the upstream signaling that regulates the above molecules, shRNA-mediated blockade of PTEN was performed. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17 and PTEN shRNAs for 24 hours followed by Western blot analysis with indicated antibodies. In the negative controls, cells were also co-transfected with p17 and respective negative controls (pGFP-V-RS and Scramble shRNA plasmids) for 24 hours. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. The protein levels were normalized to that for β-actin (panels A, B, C) or Histone H2A (panel A). The activation and inactivation folds indicated below each lane were normalized against those at 0 h or in mock control. The levels of indicated proteins at 0 h or in mock control (cell 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.g008: p17 negatively regulates ERK and cyclin D1 and positively regulates Rb through Tpr/p53/PTEN and Tpr/p53/p21 pathways.(A-B)Vero cells were transfected with pcDNA3.1-p17 or pcDNA3.1 (vector only) for 24 hours. The results of Western blot analysis of cellular fractions from the cytoplasm and nucleus (A) or whole cell lysates (B) at the indicated time points are shown. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. (C) To study whether the p17 mutant (1–118) which does not possess a NLS can affect the levels of p-ERK, cyclin D1, and CDK4 in the nucleus, vero cells were transfected with the negative control p17 mutant (1–118) for 24 hours. (D) To confirm whether PTEN is the upstream signaling that regulates the above molecules, shRNA-mediated blockade of PTEN was performed. Vero and DF-1 cells were co-transfected with pcDNA3.1-p17 and PTEN shRNAs for 24 hours followed by Western blot analysis with indicated antibodies. In the negative controls, cells were also co-transfected with p17 and respective negative controls (pGFP-V-RS and Scramble shRNA plasmids) for 24 hours. Phosphorylation and protein levels were determined by immunoblotting with the indicated antibodies. The protein levels were normalized to that for β-actin (panels A, B, C) or Histone H2A (panel A). The activation and inactivation folds indicated below each lane were normalized against those at 0 h or in mock control. The levels of indicated proteins at 0 h or in mock control (cell only) were considered 1-fold. The representative data are from three independent experiments.
Mentions: As seen from the the previous data showing that p17 activated PTEN, we next wanted to investigate whether its downstream molecules were suppressed. Nuclear PTEN is essential for tumor suppression and its nuclear import is mediated by its monoubiquitination [54]. In this study, the elevation in nuclear PTEN was found in p17-expressing cells. Consistent with a previous study [55], the increased levels of PTEN were correlated with a decrease in p-ERK and cyclin D1 levels in the nucleus in a time-dependent manner, as revealed by Western blot (Fig 8A; S3A Fig). Based on current knowledge, control of the G1/S phases of cell cycle transition is largely a matter of regulating a set of specific CDK activities. In mammalian cells, the G1/S specific CDK activities are composed of complexes between D type cyclins and either CDK4 or CDK6. A previous study has suggested that cyclin D1-CDK4 phosphorylates Rb in vivo [56]. Kitagawa and co-workers suggested that the S780 in Rb is phosphorylated during the G1 phase in a cell cycle-dependent manner and that Rb phosphorylated at S780 cannot bind to E2F-1 in vivo [56]. Importantly, this study further demonstrates that p17 negatively regulates Tpr leading to positive regulation of p53, PTEN, and p21 with the concomitant decrease in p-ERK, cyclin D1, and CDK4 levels in both Vero and DF-1 cells in a time-dependent manner, thereby resulting in Rb (S780) dephosphorylation/activation (Fig 8A and 8B; S3A and S3B Figs). The level of E2F-1 was not altered. In addition, a dramatic reduction in the cyclin D1 level in the cytoplasm and the nucleus was seen in p17-transfected cells (Fig 8A; S3A Fig). The levels of p-ERK, cyclin D1, CDK 4, and p-Rb were reversed in p17 (1–118)-transfected cells (Fig 8C) as compared to p17 transfection. The levels of PTEN and its downstream molecules are summarized in S1 Table.

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