Suppression of Vimentin Phosphorylation by the Avian Reovirus p17 through Inhibition of CDK1 and Plk1 Impacting the G2/M Phase of the Cell Cycle.
The p17 protein of avian reovirus (ARV) causes cell cycle retardation in a variety of cell lines; however, the underlying mechanism(s) by which p17 regulates the cell cycle remains largely unknown. We demonstrate for the first time that p17 interacts with CDK1 and vimentin as revealed by reciprocal co-immunoprecipitation and GST pull-down assays. Both in vitro and in vivo studies indicated that direct interaction of p17 and CDK1/vimentin was mapped within the amino terminus (aa 1-60) of p17 and central region (aa 27-118) of CDK1/vimentin. Furthermore, p17 was found to occupy the Plk1-binding site within the vimentin, thereby blocking Plk1 recruitment to CDK1-induced vimentin phosphorylation at Ser 56. Interaction of p17 to CDK1 or vimentin interferes with CDK1-catalyzed phosphorylation of vimentin at Ser 56 and subsequently vimentin phosphorylation at Ser 82 by Plk1. Furthermore, we have identified upstream signaling pathways and cellular factor(s) targeted by p17 and found that p17 regulates inhibitory phosphorylation of CDK1 and blocks vimentin phosphorylation at Ser 56 and Ser 82. The p17-mediated inactivation of CDK1 is dependent on several mechanisms, which include direct interaction with CDK1, p17-mediated suppression of Plk1 by activating the Tpr/p53 and ATM/Chk1/PP2A pathways, and p17-mediated cdc25C degradation via an ubiquitin- proteasome pathway. Additionally, depletion of p53 with a shRNA as well as inhibition of ATM and vimentin by inhibitors diminished virus yield while Tpr and CDK1 knockdown increased virus yield. Taken together, results demonstrate that p17 suppresses both CDK1 and Plk1functions, disrupts vimentin phosphorylation, causes G2/M cell cycle arrest and thus benefits virus replication.
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
Hung-Chuan
HC
Chiu
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.
Wei-Ru
WR
Huang
Department of Medical Research, Taichung Veterans General Hospital, Taichung 402, Taiwan.
Tsai-Ling
TL
Liao
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan.
Hung-Yi
HY
Wu
The Pirbright Institute, Pirbright, United Kingdom.
Muhammad
M
Munir
Department of Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan.
Wing-Ling
WL
Shih
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.|Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan.|Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan.
Hung-Jen
HJ
Liu
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division; D049109:Cell Proliferation; D000071877:Checkpoint Kinase 1 / Q000378:metabolism; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D015536:Down-Regulation; D016195:G2 Phase; D047468:Immunoprecipitation; D008954:Models, Biological; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D030101:Orthoreovirus, Avian / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D059748:Proteolysis; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D015854:Up-Regulation; D014709:Vero Cells; D014746:Vimentin / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication; D020687:cdc25 Phosphatases / Q000378:metabolism; D000097869:Polo-Like Kinase 1
D064007:Ataxia Telangiectasia Mutated Proteins; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D028861:Nuclear Pore Complex Proteins; D010768:Phosphoserine; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D014746:Vimentin; D014764:Viral Proteins; D020687:cdc25 Phosphatases; D000097869:Polo-Like Kinase 1