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

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.

Keywords: CDK inhibitor; CDK–cyclin complexes; avian reovirus; cancer; cell cycle; cyclin; cyclin-dependent kinase (CDK); cyclin-dependent kinase 7 (CDK7); mutagenesis; p17.

Figure 1.

ARV p17 competes with cyclin B1 for CDK1 binding. A, amino acid sequence alignment of ARV p17 with cyclin B1 from different species; residues framed in black boxes are conserved. Mutated residues are indicated with asterisks. B–D, Vero cells were transfected with pcDNA3.1-p17 or infected with ARV at an MOI of 5 followed by co-immunoprecipitation with CDK1 or cyclin B1 monoclonal antibodies. 10% of the input of cell lysates served as loading control, shown in Fig. S1 (A–D). Reciprocal co-immunoprecipitation assays were performed in p17 or mutant gene-transfected Vero cells and in mock controls. Western blots (WB) of CDK1- or p17-containing p17 or CDK1 immunoprecipitates were performed. 10% of the input of cell lysates served as loading control, shown in Fig. S1 (B and C). E, mutational analysis of the binding ability of p17 and p17 mutants for CDK1. To define the binding regions for CDK1 in p17, an in vitro GST pulldown assay was carried out. Elution fractions were examined by Western blotting with the indicated antibodies (CDK1 and His). 30% of the total input of TrxA-His-17 represented the internal loading control of p17. F, ribbon diagram of CDK1; diagram of electrostatic potential surface of CDK1 structure (right). The magnified figure shows the positive charge pocket of CDK1 (designated in yellow). G–I, mutational analysis of the binding ability of p17 and p17 mutants for CDK1 and CDK1 mutants. To define the binding regions for p17 in CDK1, an in vitro GST pulldown assay was carried out. Elution fractions were examined by Western blotting with the indicated antibodies (CDK1 and His). J, mutational analysis of the binding ability of p17 for CDK1. Quantitation results of the binding abilities of p17 or p17 mutants for CDK1 and CDK1 mutants are from E and G–I. Experiments were done in duplicate. In the present study, the positions of the estimated molecular mass (kDa) for all of the target proteins in the figures are indicated. The positions of the molecular mass markers in the uncropped blots are shown in Fig. S11.

Figure 2.

Identification of CDK1 and cyclin B1 interaction sites. A, mutational analysis of the binding ability of cyclin B1 for CDK1. Quantitation results of the binding ability of cyclin B1 for CDK1 are from Fig. S2, B and C. B, different CDK1 and cyclin B1 mutants were used to assess the CDK1–cyclin B1 complex kinase activity. The kinase assay measured vimentin phosphorylation at Ser-56 as analyzed by Western blotting and quantified with ImageJ software.

Figure 3.

p17 inhibits CDK1 kinase activity by direct binding to CDK1, leading to cytoplasmic retention of CDK1. A and B, in vitro kinase assays were carried out to determine the inhibitory effect of CDK1–cyclin B1 and CDK1–cyclin A2 by p17. The kinase assay measured vimentin phosphorylation at Ser-56 as analyzed by Western blotting. The p17_D36A mutant and BSA were used as negative controls. Experiments were done in duplicate. C, the inhibitory effect of p17 on CDK1 kinase activity was assessed via preincubation of p17 with CDK1 or cyclin B1 for 30 min, followed by an in vitro kinase assay. Vimentin phosphorylation at Ser-56 was analyzed by Western blotting. D, a pcDNA3.1-GFP-p17 plasmid was used to assess the distribution of CDK1 in the cytoplasm and the nucleus of Vero cells under the fluorescence microscope. Mutation at D36A was used as a negative control. Scale bar, 10 μm. E, distribution of CDK1 in the cytoplasm and the nucleus was detected by Western blotting. All data shown represent the mean ± S.D. (error bars) calculated from three independent experiments. Protein levels were normalized to those for histone H2A or β-tubulin, markers for the cytoplasm and the nucleus, respectively. Signals in all Western blots were quantified with ImageJ software.

Figure 4.

p17 binds to the preassembled CDK1–cyclin B1 and CDK1–cyclin A2 complexes. A, a flow chart shows steps in the formation of CDK1–cyclin B1 complex. The procedures in formation of CDK1–cyclin A2 complex were the same as for the CDK1–cyclin B1 complex. B, the formation of CDK1–cyclin B1 and CDK1–cyclin A2 complexes was examined by Western blotting. C, the binding ability of p17 to the preassembled CDK1–cyclin B1 complex. The values (%) indicated below each lane were p17 interacting with CDK1 or cyclin B1. 10% of total input of CDK1, cyclin B1, and p17 represented the internal loading controls. All data shown represent the mean ± S.D. (error bars) calculated from three independent experiments. D, binding ability of p17 to the preassembled CDK1–cyclin A2, CDK1_K34A/R36A–cyclin A2, and cyclin A2–CDK1_K34A/R36A complexes was examined by Western blotting. The levels of CDK1, CDK1_K34A/R36A, and cyclin A2 were considered 100%. The values indicated below each lane (p17) were normalized against values for those in CDK1, CDK1_K34A/R36A, and cyclin A2. 10% of the total input of CDK1, CDK1_K34A/R36A, cyclin A2, and p17 represented the internal loading controls. All of the data shown represent the mean ± S.D. calculated from three independent experiments. E, the inhibitory effect of CDK1–cyclin A2 kinase activity by p17 measured using an in vitro kinase assay. The level of phosphorylated vimentin (Ser-56) was analyzed by Western blotting and quantified with ImageJ software. F, two potential models for association of p17 with the CDK1–cyclin B1 and CDK1–cyclin A2 complexes.

Figure 5.

The PSTAIRE region of CDK1 is not involved in CDK1–cyclin B1 interaction. A, GST pulldown and in vitro kinase assays were performed to determine the role of Ile-49/Arg-50 in the PSTAIRE motif of CDK1 for cyclin B1 binding. The indicated proteins were examined by Western blotting. B, mutational analysis of I49A/R50A in the PSTAIRE motif of CDK2 or I59A/R60A in the PLSTIRE motif of CDK6 for partner cyclin interaction. GST pulldown and in vitro kinase assays were performed to determine the critical amino acids in CDK1 for cyclin A2, CDK2 for cyclin A2, and CDK6 for cyclin D1 binding, examined by Western blotting. 30% of total input of TrxA-His fusion protein represented the internal loading control. C, mutational analysis of the binding ability of CDK1–cyclin B1, CDK1–cyclin A2, CDK2–cyclin A2, and CDK6–cyclin D1. Quantitations of results of the binding ability of CDK and cyclin are from A and B. D, proposed model showing a positively charged pocket in CDK1 that lies very close to but in a distinct position from the PSTAIRE region.

Figure 6.

p17 binds to CDK2, cyclins (A2 and E1), and the complexes of CDK2–cyclin A2 and CDK2/E1, leading to inhibition of CDK2 kinase activity. A, amino acid sequence alignment of ARV p17 with p21^Cip1, p27^Kip1, and p57^Kip2; residues framed in black boxes are conserved. The residues mutated in p17 are indicated with asterisks. B, mutational analysis of the binding ability of p17 for CDK2, cyclin A2, cyclin E1, and their mutants based on quantitation of results from Fig. S3 (E–H). C and D, in vitro kinase assays were carried out to determine the inhibitory effect of p17 on CDK2–cyclin A2 and CDK2–cyclin E1 complexes. The kinase assay measured Rb phosphorylation at Ser-249, analyzed by Western blotting assays. p17 mutants and BSA were used as negative controls. Signals in all Western blots were quantified with ImageJ software.

Figure 7.

p17 binds to individual CDK4 and -6, cyclin D1, and the CDK6–cyclin D1 complex. A, amino acid sequence alignment of ARV p17 with CDK inhibitors showing conserved sequences with black framed residues and mutated residues in p17 indicated with asterisks. B, mutational analysis of the binding ability of p17 for CDK6 and cyclin D1 based on quantitation of results from Fig. S6 (G and H). C, in vitro kinase assays were carried out to determine the inhibitory effect of p17 on CDK6. The kinase assay measured Rb phosphorylation at Ser-780, as analyzed by Western blotting assays. The p17 mutants (R125A and K122A) and BSA were used as negative controls. The procedure used for in vitro kinase assays is described in Fig. 1F. D, the formation of CDK6–cyclin D1 complex. E, the binding ability of p17 to the preassembled CDK6–cyclin D1 complex, similar to that described in the legend to Fig. 4A. The levels of CDK6 or cyclin D1 were considered 100%. The values (%) indicated below each lane are p17 interacting with CDK6 or cyclin D1. 10% of the total input of CDK6, cyclin D1, and p17 represented the internal loading controls. Data shown represent the mean ± S.D. calculated from three independent experiments. F, in vitro kinase assays for determination of the K_i value of p17 binding to the preassembled CDK6–cyclin D1 complex. G and H, distribution of CDK6 and cyclin D1 in the cytoplasm and the nucleus was detected by Western blotting. Mutations at K122A and R125A were used as negative controls. Data shown represent the mean ± S.D. (error bars) calculated from three independent experiments. The protein levels were normalized to those for histone H2A or β-tubulin, which served as markers for the cytoplasm and the nucleus, respectively. Signals for all blots were quantified using ImageJ software.

Figure 8.

p17 promotes interaction of p53 and cyclin H to suppress CDK7. A, Western blot (WB) analysis of CDK7– or cyclin H–containing p17 immunoprecipitates was performed. Rabbit or mouse IgG served as negative controls. Representative data from three independent experiments are shown. The -fold changes indicated below each lane were normalized against the values in the mock controls. The level of the indicated proteins in the mock control was considered 1-fold. B, Vero cells were transfected with the indicated plasmid DNA for 24 h. The expression levels of p-CDK2 (Thr-160), CDK2, and p53 were analyzed by Western blots in p17 and p53 shRNA co-transfected cells. The protein levels were normalized to levels for β-actin. Signals for all blots were quantified using ImageJ software. C, illustration of the ARV p17–mediated activation of p53 to disassociate the CDK7–cyclin H complex, reducing CDK2 phosphorylation at Thr-160.

Figure 9.

The effects of p17 on CDKs in cancer cell lines. A, co-immunoprecipitation assays were performed in p17-transfected HeLa and SW620 cells and in mock controls using antibodies against p17, CDK, and cyclins. B, in reciprocal co-immunoprecipitation experiments, the interaction of p17 and CDKs was further examined in p17-transfected HeLa cells by Western blotting (WB) of p17 immunoprecipitates. C, the levels of CDKs, p-Rb (Ser-249 and Ser-780), and p-vimentin (Ser-56) were examined in p17-transfected HeLa and SW620 and mock control cells. D, MG132 (2.5 μm) was used to treat mock, ARV-infected, and p17-transfected cells. The protein levels of CDK2 and CDK4 were examined by Western blotting. E, to examine whether CDK transcription was down-regulated by p17, mRNA levels of CDKs in pcDNA3.1-p17–transfected HeLa cells were examined at the indicated time points. HeLa cells were transfected with pcDNA3.1-p17, and transfected cells were collected at 0, 12, or 24 h, followed by semiquantitative RT-PCR for analysis of CDK genes. After electrophoretic analysis, PCR products were stained with ethidium bromide. Data in the graph represent the mean ± S.D. (error bars) calculated from three independent experiments. Signals for all blots were quantified using ImageJ software.

Figure 10.

Exogenous expression of p17 significantly enhances virus replication in ARV-infected cells. A, 4-well cell culture plates were transfected with p17 for 6 h followed by infection with different MOIs of ARV. The supernatants of each well were harvested at 24 hpi for viral titration. B, 4-well cell culture plates were transfected with pcDNA3.1-p17 or mutants for 6 h, followed by infection with an MOI of 0.1. The supernatants of each well were harvested at 24 hpi for viral titration. The expression levels of p17, p17 mutants, and the empty pcDNA3.1 control for every condition were examined by Western blotting. Error bars, S.D.

Figure 11.

p17 results in cell growth inhibition and cell cycle retardation in various mammalian, avian, and cancer cell lines. A, the growth curve of various cell lines transfected with pcDNA3.1, p17-pcDNA3.1, or p17 mutant genes. The data represent the average of triplicate plates with S.D. (error bars) indicated. The x axis represents the post-transfection time of incubation. The viable cells were counted with a hemocytometer in the presence of trypan blue and are represented on the y axis. B, p17 causes cell cycle retardation in various mammalian, avian, and cancer cell lines. Cells were transfected for 18 h with constructs after serum deprivation for 54 h. As shown in Fig. S10C, the percentages of cells accumulating in each phase of the cell cycle at different time points were analyzed. B was derived from the results of Fig. S10C. The x axis represents the time period of incubation post-transfection, whereas cell cycle number is represented on the y axis. C, 1 × 10⁷ A549 lung cancer cells were injected subcutaneously into BALB/c nude (nu/nu) mice and monitored for tumorigenesis in vivo. A549 cancer cells in nude (nu/nu) mice were allowed to grow for 17 days, followed by gene gun transformation with pcDNA3.1-p17 and pcDNA3.1 plasmids, respectively. n = 3 for each group.