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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

The NLS within p17 directly interacts with Tpr.(A) To map the region within p17 that was involved in Tpr binding, a series of truncated versions of Flag-tagged p17 constructs were established. Schematic representation of p17 deletion mutants is shown. The ability of the p17 and truncated p17 mutants to interact with Tpr is shown on the right hand side of the panel. +, strong binding;-, no binding. (B-C) Cellular lysates from Vero cells transfected with Flag-tagged p17 deletion vectors were immunoprecipitated with anti-Tpr and anti-Flag antibodies, respectively. Following immunoblotting analysis, Tpr and Flag-tagged p17 deletion proteins (1–118 and 27–146) (panel B) and p17 (119–146) (panel C) were detected by using Tpr and Flag antibodies. (D) Immunoprecipitation of Tpr by using an anti-Tpr antibody was performed. The immunoprecipitated Tpr proteins were separated by SDS-PAGE followed by Western blot assay using an anti-Tpr antibody (lower panel). Purified TrxA-His-p17 fusion protein was analyzed by SDS-PAGE followed by Western blot assay using an anti-p17 antibody (upper panel). Rabbit IgG was used as a negative control. (E) In vitro binding assays using a synthetic peptide (His-p17-NLS, His6-IAAKRGRQLD) and purified TrxA-His-p17 fusion protein were performed. The synthetic peptide His-p17-NLS (lower panel) and purified TrxA-His-p17 fusion protein (upper panel) were then subjected to analysis for their binding abilities to Tpr, as revealed by Western blot and dot blot assays, as indicated. The representative data are from three independent experiments.
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pone.0133699.g002: The NLS within p17 directly interacts with Tpr.(A) To map the region within p17 that was involved in Tpr binding, a series of truncated versions of Flag-tagged p17 constructs were established. Schematic representation of p17 deletion mutants is shown. The ability of the p17 and truncated p17 mutants to interact with Tpr is shown on the right hand side of the panel. +, strong binding;-, no binding. (B-C) Cellular lysates from Vero cells transfected with Flag-tagged p17 deletion vectors were immunoprecipitated with anti-Tpr and anti-Flag antibodies, respectively. Following immunoblotting analysis, Tpr and Flag-tagged p17 deletion proteins (1–118 and 27–146) (panel B) and p17 (119–146) (panel C) were detected by using Tpr and Flag antibodies. (D) Immunoprecipitation of Tpr by using an anti-Tpr antibody was performed. The immunoprecipitated Tpr proteins were separated by SDS-PAGE followed by Western blot assay using an anti-Tpr antibody (lower panel). Purified TrxA-His-p17 fusion protein was analyzed by SDS-PAGE followed by Western blot assay using an anti-p17 antibody (upper panel). Rabbit IgG was used as a negative control. (E) In vitro binding assays using a synthetic peptide (His-p17-NLS, His6-IAAKRGRQLD) and purified TrxA-His-p17 fusion protein were performed. The synthetic peptide His-p17-NLS (lower panel) and purified TrxA-His-p17 fusion protein (upper panel) were then subjected to analysis for their binding abilities to Tpr, as revealed by Western blot and dot blot assays, as indicated. The representative data are from three independent experiments.

Mentions: The above specific binding led us to further define the region within p17 for Tpr binding by using a series of Flag-tagged p17 deletion mutants. Schematic representation of p17 deletion mutants is shown in Fig 2A. The C-terminally truncated p17 mutant (1–118) and N-terminally truncated p17 mutants (27–146 and 119–146) were constructed. Cellular lysates from transfection with Flag-tagged p17 deletion vectors were immunoprecipitated with anti-Tpr and anti-Flag antibodies, respectively. Following immunoblotting assays, the Flag-tagged deleted p17 were detected (Fig 2B and 2C). The results presented in Fig 2C revealed that the deletions in p17-(27–146) and p17-(119–146), both of which still contain the NLS, did not abolish their interaction with Tpr. In contrast, the ability to interact with Tpr was abolished when the NLS within the C-terminal region of p17 (1–118) were removed [36]. To assess the consequences, we further examined whether the the C-terminal portion of p17 bearing NLS is required for interaction. Our results reveal that the NLS within p17 is critical for Tpr interaction, as revealed by reciprocal coi-mmunoprecipitation assay.


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)

The NLS within p17 directly interacts with Tpr.(A) To map the region within p17 that was involved in Tpr binding, a series of truncated versions of Flag-tagged p17 constructs were established. Schematic representation of p17 deletion mutants is shown. The ability of the p17 and truncated p17 mutants to interact with Tpr is shown on the right hand side of the panel. +, strong binding;-, no binding. (B-C) Cellular lysates from Vero cells transfected with Flag-tagged p17 deletion vectors were immunoprecipitated with anti-Tpr and anti-Flag antibodies, respectively. Following immunoblotting analysis, Tpr and Flag-tagged p17 deletion proteins (1–118 and 27–146) (panel B) and p17 (119–146) (panel C) were detected by using Tpr and Flag antibodies. (D) Immunoprecipitation of Tpr by using an anti-Tpr antibody was performed. The immunoprecipitated Tpr proteins were separated by SDS-PAGE followed by Western blot assay using an anti-Tpr antibody (lower panel). Purified TrxA-His-p17 fusion protein was analyzed by SDS-PAGE followed by Western blot assay using an anti-p17 antibody (upper panel). Rabbit IgG was used as a negative control. (E) In vitro binding assays using a synthetic peptide (His-p17-NLS, His6-IAAKRGRQLD) and purified TrxA-His-p17 fusion protein were performed. The synthetic peptide His-p17-NLS (lower panel) and purified TrxA-His-p17 fusion protein (upper panel) were then subjected to analysis for their binding abilities to Tpr, as revealed by Western blot and dot blot assays, as indicated. The representative data are from three independent experiments.
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Related In: Results  -  Collection

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pone.0133699.g002: The NLS within p17 directly interacts with Tpr.(A) To map the region within p17 that was involved in Tpr binding, a series of truncated versions of Flag-tagged p17 constructs were established. Schematic representation of p17 deletion mutants is shown. The ability of the p17 and truncated p17 mutants to interact with Tpr is shown on the right hand side of the panel. +, strong binding;-, no binding. (B-C) Cellular lysates from Vero cells transfected with Flag-tagged p17 deletion vectors were immunoprecipitated with anti-Tpr and anti-Flag antibodies, respectively. Following immunoblotting analysis, Tpr and Flag-tagged p17 deletion proteins (1–118 and 27–146) (panel B) and p17 (119–146) (panel C) were detected by using Tpr and Flag antibodies. (D) Immunoprecipitation of Tpr by using an anti-Tpr antibody was performed. The immunoprecipitated Tpr proteins were separated by SDS-PAGE followed by Western blot assay using an anti-Tpr antibody (lower panel). Purified TrxA-His-p17 fusion protein was analyzed by SDS-PAGE followed by Western blot assay using an anti-p17 antibody (upper panel). Rabbit IgG was used as a negative control. (E) In vitro binding assays using a synthetic peptide (His-p17-NLS, His6-IAAKRGRQLD) and purified TrxA-His-p17 fusion protein were performed. The synthetic peptide His-p17-NLS (lower panel) and purified TrxA-His-p17 fusion protein (upper panel) were then subjected to analysis for their binding abilities to Tpr, as revealed by Western blot and dot blot assays, as indicated. The representative data are from three independent experiments.
Mentions: The above specific binding led us to further define the region within p17 for Tpr binding by using a series of Flag-tagged p17 deletion mutants. Schematic representation of p17 deletion mutants is shown in Fig 2A. The C-terminally truncated p17 mutant (1–118) and N-terminally truncated p17 mutants (27–146 and 119–146) were constructed. Cellular lysates from transfection with Flag-tagged p17 deletion vectors were immunoprecipitated with anti-Tpr and anti-Flag antibodies, respectively. Following immunoblotting assays, the Flag-tagged deleted p17 were detected (Fig 2B and 2C). The results presented in Fig 2C revealed that the deletions in p17-(27–146) and p17-(119–146), both of which still contain the NLS, did not abolish their interaction with Tpr. In contrast, the ability to interact with Tpr was abolished when the NLS within the C-terminal region of p17 (1–118) were removed [36]. To assess the consequences, we further examined whether the the C-terminal portion of p17 bearing NLS is required for interaction. Our results reveal that the NLS within p17 is critical for Tpr interaction, as revealed by reciprocal coi-mmunoprecipitation assay.

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