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. 2006 Feb;80(4):2045-50.
doi: 10.1128/JVI.80.4.2045-2050.2006.

cdc2/cyclin B1-dependent phosphorylation of EBNA2 at Ser243 regulates its function in mitosis

Wei Yue  1 , Julia Shackelford, Joseph S Pagano
Affiliations

cdc2/cyclin B1-dependent phosphorylation of EBNA2 at Ser243 regulates its function in mitosis

Wei Yue et al. J Virol. 2006 Feb.

Abstract

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) transactivates EBV genes in latently infected B cells. We have shown that mitotic hyperphosphorylation of EBNA2 suppresses its ability to transactivate the latent membrane protein 1 (LMP1) promoter. In this follow-up study, we identify EBNA2 Ser243 as a phosphorylation site for mitotic cdc2/cyclin B1 kinase. Mutation at Ser243, which mimics constitutive phosphorylation of the protein, decreases endogenous levels of both LMP1 and EBNA2. Moreover, mutation at Ser243 reduces the ability of EBNA2 to transactivate Cp, the promoter for all six EBV EBNA genes. Our data implicate EBNA2 Ser243 as a cdc2/cyclin B1 site of phosphorylation important for EBNA2's cotranscriptional function in mitosis.

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Figures

FIG. 1.
FIG. 1.
Phosphorylation of cdc2 site Ser243 in vitro and in vivo in mitosis. (A) Phosphorylation of purified GST-EBNA2 185-313aa (GST-E2), GST-EBNA2 185-313aa Ser243A (GST-E2 S243A) or GST in the presence (+) or absence (−) of cdc2/cyclin B1 kinase was carried out. The reaction mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4 to 20% gradient gels). After Coomassie bright blue (CBB) staining, gels were exposed on a PhosphorImager. (B) Transiently expressed wild-type EBNA2 (wt) or EBNA2 S243A in HeLa cells were immunoprecipitated by EBNA2 antibody and used as substrates for cdc2/cyclin B1 kinase assays. Immunocomplexes were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto membrane; phosphorylation of EBNA2 was detected by autoradiography (top panel). Equal protein levels of EBNA2 were confirmed by immunoblotting (bottom panel). (C) Migration of wild-type EBNA2 and EBNA2 Ser243 in different phases of the cell cycle. HeLa cells were synchronized at the G1/S border by double-thymidine block. S- and G1-phase cells were collected 6 and 24 h after release from thymidine, and M-phase cells were collected by vigorous shaking-off at 10 h after release.
FIG. 2.
FIG. 2.
Induction of endogenous LMP1 expression in P3HR1 cells by EBNA2. Wild-type EBNA2, EBNA2 Ser243D, or EBNA2 Ser243E were transiently expressed in P3HR1 cells through electroporation. Transfection with empty vector was used as control. (A) Whole-cell lysates were subjected to immunoblotting for LMP1 and EBNA2 48 h after transfection. β-Actin was used as a loading control. (B) Transfected cells were selected with anti-CD4 magnetic beads, and total RNA was extracted and subjected to RPA for LMP1 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) RNAs. Yeast RNA was used as negative and X50-7 RNA positive controls. A histogram quantifying LMP1 mRNA signals by normalization with GAPDH mRNA signals is shown. (C) Immunoblotting of EBNA2 in P3HR1 cells after nuclear and cytoplasm separation 48 h posttransfection. Lanes 1, 2, 3, and 4 contained EBNA2 Ser243E, EBNA2 Ser243D, wild-type EBNA2, and vector, respectively. Lamin B is used as a nuclear marker.
FIG. 3.
FIG. 3.
Phosphorylation at Ser243 suppresses EBNA2 transactivation of the EBV BamHI C promoter, Cp. HeLa cells were cotransfected with reporter plasmid pLuc-Cp and pRL-TK, which encodes the Renilla luciferase gene in addition to empty vector or EBNA2 expression vector encoding wild-type EBNA2 (EBNA2 wt) or EBNA2 Ser243D. At 24 h after transfection, cells were arrested in M phase by nocodazole (M); untreated asynchronously growing cells served as a control (Asy). Luciferase assays were performed to evaluate promoter activity. In each phase, transactivation of Cp by EBNA2 is normalized to the vector control. (A) Decreased transactivation of Cp by EBNA2 in M phase. *, P < 0.01 versus control. (B) Comparison of transactivation of Cp by wild-type EBNA2, EBNA2 Ser243A and EBNA2 Ser243D. Each datum point (A and B) represents the average of three independent experiments done in triplicate. Error bars represent the means ± the standard deviation.
FIG. 4.
FIG. 4.
Decreased endogenous steady-state EBNA2 mRNA levels in M-phase-arrested cells. (A) RPA was performed on total RNA from asynchronously growing (Asy) and M-phase-arrested (M) LCL-1 cells with GAPDH and EBNA2 probe. Yeast RNA or total RNA from DG75 cells were used as negative controls. A representative result from three independent experiments is shown. (B) Relative mRNA levels of EBNA2 were analyzed by normalizing EBNA2 mRNA levels to the GAPDH mRNA level with a PhosphorImager. Each datum point represents the average of three independent experiments. Error bars represent the means ± standard deviation (*, P < 0.01 versus Asy control).
FIG. 5.
FIG. 5.
Alignment of the type 1 (B95-8), type 2 (AG876), baboon LCV and rhesus LCV EBNA2 amino acid sequences from amino acids 230 to 328. Clusters of amino acids CR5 and CR6 (32) that are conserved among all four EBNA2 proteins are indicated (boxed), as is the positionally conserved, cdc2 consensus phosphorylation motif (boxed). The putative cdc2 phosphorylation sites are marked with an asterisk.
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