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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

Identification of potential cellular factors that interact with p17.(A)Vero cells were transfected with pcDNA3.1-p17 plasmid for 24 hours, followed by a co-immunoprecipatation assay by p17 antibody. Cellular proteins co-immunoprecipated by p17 antibody were analyzed by SDS-PAGE. Lane 1: protein marker; lane 2: Co-immunoprecipated proteins by p17 antibody. (B) Reciprocal co-immunoprecipitation assays in both ARV-infected and p17-transfected cells were performed with either p17 or Tpr antibodies.
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pone.0133699.g001: Identification of potential cellular factors that interact with p17.(A)Vero cells were transfected with pcDNA3.1-p17 plasmid for 24 hours, followed by a co-immunoprecipatation assay by p17 antibody. Cellular proteins co-immunoprecipated by p17 antibody were analyzed by SDS-PAGE. Lane 1: protein marker; lane 2: Co-immunoprecipated proteins by p17 antibody. (B) Reciprocal co-immunoprecipitation assays in both ARV-infected and p17-transfected cells were performed with either p17 or Tpr antibodies.

Mentions: To identify potential cellular factors that specifically interact with p17, Vero cells were transfected with a plasmid containing p17 (pcDNA3.1-p17) for 24 hours and followed by a co-immunoprecipatation assay with the p17 antibody. To obtain large amounts of p17-coimmunoprecipatated proteins for mass spectrometry analysis, Vero cells in fifteen 6-cm cell culture dishes were harvested and concentrated. Interestingly, several cellular proteins were coimmunoprecipated by the p17 antibody, as revealed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (Fig 1A). Three host factors (necleoporin Tpr, lamin A/C and vimentin) that coimmunoprecipated with p17 were then identified by mass spectrometry analysis. To further investigate the interplay between p17 and Tpr, Tpr with molecular mass of approximately 267 kDa that specifically coimmunoprecipated with p17 (Fig 1B) was further studied. To rule out the possibility of overexpression artifacts, reciprocal co-immunoprecipation assays were performed in both ARV-infected and p17-transfected cells as well as in mock controls. Reciprocally, p17 could also be pulled down in both ARV-infected and p17-transfected cells when an antibody against Tpr was used for immunoprecipitation (Fig 1B).


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)

Identification of potential cellular factors that interact with p17.(A)Vero cells were transfected with pcDNA3.1-p17 plasmid for 24 hours, followed by a co-immunoprecipatation assay by p17 antibody. Cellular proteins co-immunoprecipated by p17 antibody were analyzed by SDS-PAGE. Lane 1: protein marker; lane 2: Co-immunoprecipated proteins by p17 antibody. (B) Reciprocal co-immunoprecipitation assays in both ARV-infected and p17-transfected cells were performed with either p17 or Tpr antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526660&req=5

pone.0133699.g001: Identification of potential cellular factors that interact with p17.(A)Vero cells were transfected with pcDNA3.1-p17 plasmid for 24 hours, followed by a co-immunoprecipatation assay by p17 antibody. Cellular proteins co-immunoprecipated by p17 antibody were analyzed by SDS-PAGE. Lane 1: protein marker; lane 2: Co-immunoprecipated proteins by p17 antibody. (B) Reciprocal co-immunoprecipitation assays in both ARV-infected and p17-transfected cells were performed with either p17 or Tpr antibodies.
Mentions: To identify potential cellular factors that specifically interact with p17, Vero cells were transfected with a plasmid containing p17 (pcDNA3.1-p17) for 24 hours and followed by a co-immunoprecipatation assay with the p17 antibody. To obtain large amounts of p17-coimmunoprecipatated proteins for mass spectrometry analysis, Vero cells in fifteen 6-cm cell culture dishes were harvested and concentrated. Interestingly, several cellular proteins were coimmunoprecipated by the p17 antibody, as revealed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (Fig 1A). Three host factors (necleoporin Tpr, lamin A/C and vimentin) that coimmunoprecipated with p17 were then identified by mass spectrometry analysis. To further investigate the interplay between p17 and Tpr, Tpr with molecular mass of approximately 267 kDa that specifically coimmunoprecipated with p17 (Fig 1B) was further studied. To rule out the possibility of overexpression artifacts, reciprocal co-immunoprecipation assays were performed in both ARV-infected and p17-transfected cells as well as in mock controls. Reciprocally, p17 could also be pulled down in both ARV-infected and p17-transfected cells when an antibody against Tpr was used for immunoprecipitation (Fig 1B).

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