Abstract
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 151LAVXDVDA(E/D)DGADPN165 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, 125RXL127 Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 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 in vivo tumorigenesis assay also showed a significant reduction in tumor size.
Highlights
The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein
This study provides novel insights into p17-modulated suppression of cyclins, Cyclin-dependent kinases (CDKs) activity, complex formation, and the cell cycle in mammalian, avian, and cancer cell lines
In addition to activation of p21 (CIP/KIP family) [8, 11], p17 has broader inhibitory effects on cell cycle CDK kinases in several cell lines compared with the NK4 and CIP/KIP families, suggesting that the Avian reoviruses (ARVs) p17 protein represents a new class of CDK inhibitor
Summary
A previous study suggested that a cyclin box in cyclin B1 is involved in CDK1 binding [15]. The second model indicates that inhibition of CDK– cyclin A2 kinase activity occurs through direct binding of p17 to CDK1, cyclin A2, or the complexes. In contrast to p21, which promotes the formation of the CDK6 – cyclin D1 complex and promotes increased kinase activity [28], p17 exhibits weak binding ability to the preassembled CDK6 – cyclin D1 complex (less than 50%; Fig. 7, D and E), and the Ki value for inhibition of CDK6 – cyclin D1 by p17 was only 500 nM (Fig. 7F). The previous and current studies suggest that p17-modulated inhibitions of CDK1 function by direct binding to CDK1 to suppress the formation of CDK1– cyclin B1 complex and by suppression of both Plk and CDC25C to cause inhibitory phosphorylation of CDK1 [12]. An in vivo tumorigenesis assay was carried out to evaluate effects of p17 on the growth of A549 cancer cells (a lung cancer cell line), which showed a significant reduction in tumor size (Fig. 11C)
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