Suppression of Vimentin Phosphorylation by the Avian Reovirus p17 through Inhibition of CDK1 and Plk1 Impacting the G2/M Phase of the Cell Cycle

Wang

et al. 2019

J Virol

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Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19 LSLRELAI 26 of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/Cmodulated nucleocytoplasmic shuttling of the ARV p17 protein.IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein. RESULTSCellular proteins hnRNP A1 and lamin A/C coimmunoprecipatate with p17. As shown previously, we identified several cellular proteins, such as nucleoporin Tpr, vimentin, and lamin A/C, that interact with the ARV p17 protein (27,28,30). To explore whether and how cellular proteins are involved in mediating nucleocytoplasmic shut-Chiu et al. FIG 1 p17 interacts with hnRNP A1. (A) Identification of poten...

Section: Resultsmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17

Wang

et al. 2019

J Virol

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“…The ARV p17 protein acts as a positive regulator by stabilizing PTEN by phosphorylation of its cytoplasmic form and by elevating Rak-PTEN interaction to prevent it from E3 ligase NEDD4-1 targeting (11). p17 suppresses CDK1 function by suppressing both Plk1 and CDC25C to cause inhibitory phosphorylation of CDK1 (12). More recently, we found that the p17 and A proteins of ARV act cooperatively to down-regulate Akt by suppressing both mTORC2 and CDK2 and up-regulating proteasome PSMB6 (13).…”

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confidence: 84%

Mechanistic insights into avian reovirus p17-modulated suppression of cell cycle CDK–cyclin complexes and enhancement of p53 and cyclin H interaction

Journal of Biological Chemistry

Liao

et al. 2018

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The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK-cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK-cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53-cyclin H interaction. p17 binds to CDK-cyclin except for CDK1-cyclin B1 and CDK7-cyclin H complexes. We have determined that the negatively charged LAVDVDA(E/D)DGADPN motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1-cyclin B1, but it is required for other CDK-cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, RL Sequence and mutagenic analyses of p17 indicated that a WFD motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An tumorigenesis assay also showed a significant reduction in tumor size.

“…Data are expressed as mean ± SD of at least three independent experiments 23, 24 . In all tests, p < 0.05 was considered statistically significant.…”

Section: Methodsmentioning

confidence: 99%

Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6

Chi

et al. 2017

Sci Rep

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Although we have shown that avian reovirus (ARV) p17-mediated inhibition of Akt leads to induction of autophagy, the precise mechanisms remain largely unknown. This study has identified a specific mechanism by which ARV coordinately regulates the degradation of ribosomal proteins by p17-mediated activation of E3 ligase MDM2 that targets ribosomal proteins and by σA-mediated upregulation of proteasome PSMB6. In addition to downregulating ribosomal proteins, p17 reduces mTORC2 assembly and disrupts mTORC2-robosome association, both of which inactivate mTORC2 leading to inhibition of Akt phosphorylation at S473. Furthermore, we discovered that p17 binds to and inhibits the CDK2/cyclin A2 complex, further inhibiting phosphorylation of Akt S473. The negative effect of p17 on mTORC2 assembly and Akt phosphorylation at S473 is reversed in cells treated with insulin or overexpression of CDK2. The carboxyl terminus of p17 is necessary for interaction with CDK2 and for induction of autophagy. Furthermore, p17-mediated upregulation of LC3-II could be partially reversed by overexpression of CDK2. The present study provides mechanistic insights into cooperation between p17 and σA proteins of ARV to negatively regulate Akt by downregulating complexes of mTORC2 and CDK2/cyclin A2 and upregulating PSMB6, which together induces autophagy and cell cycle arrest and benefits virus replication.