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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 PI3K/AKT/mTOR signaling pathway.(A-B) Vero cells were transfected with pcDNA3.1-p17 and pcDNA3.1 (vector only) plasmid, respectively for 24 hours. Whole cell lysates were collected at the indicated time points, and the levels of PI3K and its downstream molecules were examined by Western blot assay with the indicated antibodies. (B)Vero and DF-1 cells were co-transfected with both pcDNA3.1-p17 and p53 shRNAs for 24 hours, followed by Western blot analysis with indicated antibodies. Cells were also co-transfected with pCDNA3.1- p17 and respective negative controls (scrambled shRNAs and pGFP-V-RS vector) for 24 hours. (C) To study whether the negative control p17 mutant (1–118) can affect the levels of p-PTEN, p-AKT, p-mTOR, and LC3-II, vero cells were transfected with the p17 mutant (1–118) plasmid for 24 hours. Similar results were obtained from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins at 0 h or mock were considered 1-fold.
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pone.0133699.g009: p17 negatively regulates PI3K/AKT/mTOR signaling pathway.(A-B) Vero cells were transfected with pcDNA3.1-p17 and pcDNA3.1 (vector only) plasmid, respectively for 24 hours. Whole cell lysates were collected at the indicated time points, and the levels of PI3K and its downstream molecules were examined by Western blot assay with the indicated antibodies. (B)Vero and DF-1 cells were co-transfected with both pcDNA3.1-p17 and p53 shRNAs for 24 hours, followed by Western blot analysis with indicated antibodies. Cells were also co-transfected with pCDNA3.1- p17 and respective negative controls (scrambled shRNAs and pGFP-V-RS vector) for 24 hours. (C) To study whether the negative control p17 mutant (1–118) can affect the levels of p-PTEN, p-AKT, p-mTOR, and LC3-II, vero cells were transfected with the p17 mutant (1–118) plasmid for 24 hours. Similar results were obtained from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins at 0 h or mock were considered 1-fold.

Mentions: As its lipid phosphatase activity by converting PIP3 to PIP2, PTEN prevents AKT activation. Therefore, we next examined the effect of p17 on promoting β-arrestin-mediated PTEN regulation by looking at p-AKT levels. The plasma membrane-associated PTEN level (Fig 6B) was elevated with concomitant PDK-1 and AKT dephosphorylation/inactivation in total cell lysate (Fig 9A; S4 Fig) in a time-dependent manner. An earlier study has suggested that mTORC2 phosphorylates AKT at S473 in its C-terminal hydrophobic motif, which in conjunction with PDK1-regulated phosphorylation at T308, drives full activation of AKT [57]. Our results reveal that p17 dramatically reduces phosphorylation of AKT at T308 and S473 in total cell lysate (Fig 9A; S4 Fig) as compared to the negative control (vector only). It is important to note that although p21 phosphorylation at T145 is shown to be dependent on AKT (Fig 4C) and that at least eighty five percent of p-AKT levels are reduced by p17 (Fig 9A and 9C), the elevated levels of p-p21 in the nucleus in both ARV-infected and p17-transfected cells (Fig 4C, lanes 2–3) are likely due to p21 phosphorylation by the residual activated AKT and 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 negatively regulates PI3K/AKT/mTOR signaling pathway.(A-B) Vero cells were transfected with pcDNA3.1-p17 and pcDNA3.1 (vector only) plasmid, respectively for 24 hours. Whole cell lysates were collected at the indicated time points, and the levels of PI3K and its downstream molecules were examined by Western blot assay with the indicated antibodies. (B)Vero and DF-1 cells were co-transfected with both pcDNA3.1-p17 and p53 shRNAs for 24 hours, followed by Western blot analysis with indicated antibodies. Cells were also co-transfected with pCDNA3.1- p17 and respective negative controls (scrambled shRNAs and pGFP-V-RS vector) for 24 hours. (C) To study whether the negative control p17 mutant (1–118) can affect the levels of p-PTEN, p-AKT, p-mTOR, and LC3-II, vero cells were transfected with the p17 mutant (1–118) plasmid for 24 hours. Similar results were obtained from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins at 0 h or mock were considered 1-fold.
© Copyright Policy
Related In: Results  -  Collection

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pone.0133699.g009: p17 negatively regulates PI3K/AKT/mTOR signaling pathway.(A-B) Vero cells were transfected with pcDNA3.1-p17 and pcDNA3.1 (vector only) plasmid, respectively for 24 hours. Whole cell lysates were collected at the indicated time points, and the levels of PI3K and its downstream molecules were examined by Western blot assay with the indicated antibodies. (B)Vero and DF-1 cells were co-transfected with both pcDNA3.1-p17 and p53 shRNAs for 24 hours, followed by Western blot analysis with indicated antibodies. Cells were also co-transfected with pCDNA3.1- p17 and respective negative controls (scrambled shRNAs and pGFP-V-RS vector) for 24 hours. (C) To study whether the negative control p17 mutant (1–118) can affect the levels of p-PTEN, p-AKT, p-mTOR, and LC3-II, vero cells were transfected with the p17 mutant (1–118) plasmid for 24 hours. Similar results were obtained from three independent experiments. The protein levels were normalized to those for β-actin.The activation and inactivation folds indicated below each lane were normalized against those at 0 h or mock. The levels of indicated proteins at 0 h or mock were considered 1-fold.
Mentions: As its lipid phosphatase activity by converting PIP3 to PIP2, PTEN prevents AKT activation. Therefore, we next examined the effect of p17 on promoting β-arrestin-mediated PTEN regulation by looking at p-AKT levels. The plasma membrane-associated PTEN level (Fig 6B) was elevated with concomitant PDK-1 and AKT dephosphorylation/inactivation in total cell lysate (Fig 9A; S4 Fig) in a time-dependent manner. An earlier study has suggested that mTORC2 phosphorylates AKT at S473 in its C-terminal hydrophobic motif, which in conjunction with PDK1-regulated phosphorylation at T308, drives full activation of AKT [57]. Our results reveal that p17 dramatically reduces phosphorylation of AKT at T308 and S473 in total cell lysate (Fig 9A; S4 Fig) as compared to the negative control (vector only). It is important to note that although p21 phosphorylation at T145 is shown to be dependent on AKT (Fig 4C) and that at least eighty five percent of p-AKT levels are reduced by p17 (Fig 9A and 9C), the elevated levels of p-p21 in the nucleus in both ARV-infected and p17-transfected cells (Fig 4C, lanes 2–3) are likely due to p21 phosphorylation by the residual activated AKT and 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