Vpr promotes cytoplasmic compartmentalization of Cdc25 through serine/threonine phosphorylation of Cdc25. The wild-type SP223 strain (A) and the RE007 (B) yeast strain, which is a derivative of the SP223 containing a single integrated copy of the vpr gene under the control of the nmt1 promoter, were transformed with pREP41x-GFP, pREP41x-GFP-cdc25, or pREP41x-GFP-cdc25(9A). To visualize the subcellular localization of Cdc25, these strains were grown in liquid selective medium in the absence of thiamine for 20 h to induce gene expression. (Ba) All of the cells were collected 20 h after gene induction and visualized microscopically. Cdc25 was detected by the emission of green fluorescence, and nuclear DNA was stained with DAPI. Bar, 10 μm. (Bb) Immunoblot analysis was performed in the test strains to confirm the absence or expression of vpr in the wild type or the RE007 strains after transformation with the plasmids of pREP41x-GFP, pREP41x-GFP-cdc25, or pREP41x-GFP-cdc25(9A), labeled 1, 2, and 3, respectively. (C) The RE109 yeast strain, which is derived from RE007 strain carrying an integrated copy of the cdc25-gfp gene, was grown in liquid culture for 20 h in the presence (vpr-off) or in the absence (vpr-on) of thiamine to induce vpr gene expression. The cdc25-gfp gene is constitutively expressed under its native promoter (7). Immunoblot analysis was carried out and confirmed the expression of vpr in the RE109 strain (data not shown). For all of the immunoblot analyses, vpr-repressing and vpr-expressing cells were collected and lysed 20 h after gene induction. Equal amounts of proteins (25 μg) were loaded onto a 10 to 20% gradient SDS-PAGE gel. Vpr was visualized with anti-Vpr (dilution, 1/500). (D) Vpr promotes protein degradation of Cdc25. Wild-type (SP223) and RE007 yeast strains transformed with pREP41x-GFP-Cdc25 were grown in liquid culture for 24 h in the presence or absence of thiamine to induce gene expression. Cell fractionation was performed to obtain cytoplasmic (C) and nuclear (N) extracts from the cells. A Western blot analysis was carried out to detect Vpr and Cdc25. G.I., gene induction.

Fission yeast cells defective in DNA damage or DNA replication checkpoints do not affect Vpr-mediated nuclear export of Cdc25. The rad3-136 mutation blocks nuclear export of Cdc25 induced by cisplatin (A) but not that induced by Vpr (B). The wild-type (Q2016) and rad3-136 (RE285) fission yeast strains were grown in liquid culture for 20 h in the presence of thiamine. When indicated, cisplatin was added to the culture at a final concentration of 0.5 mM, and the cell size and subcellular location of Cdc25 were determined 4.5 h after treatment. The wild-type (RE109) and rad3-136 (RE285) fission yeast strains were grown and assayed the same way as described above. Bar, 10 μm.

Vpr induces cell cycle G₂ arrest through direct interaction with Srk1. (A) Coexpression of vpr and srk1 showed no additive effect on Vpr-induced cell elongation. (a) Overexpression of srk1 induces cell cycle G₂ arrest with cell elongation. The wild-type (SP223) yeast strain was transformed with pREP1-srk1(6HisHA) or pREP1-srk1(K153A)(6HisHA) and grown for 27 h in the minimal selective EMM liquid culture either in the presence or in the absence of thiamine to induce gene expression. The average cell length and standard derivation was measured on at least 100 cells by using the OpenLab software and based on three independent experiments. (b) Coexpression of vpr and srk1 showed no additive effect on Vpr-induced cell elongation. The RE076 fission yeast strain, which carries a single integrated copy of F34Ivpr gene (13), was transformed with pREP1-srk1(6HisHA) or pREP1-srk1(K153A)(6HisHA) and grown and assayed as described for panel Aa. Immunoblot analysis was performed to confirm the expression of srk1(6HisHA), srk1(K153A)(6HisHA), and vpr in the SP223 and RE076 strains transformed with pREP1-srk1(6HisHA) or pREP1-srk1(K153A)(6HisHA). The same levels of Vpr or Srk1 proteins were expressed in each of these samples (data not shown). (B) Deletion of the srk1 gene abolished Vpr-induced cell elongation. The srk1⁺ (RE076) and Δsrk1 (SH2) fission yeast strains were grown for 20 h in the minimal selective EMM liquid culture in the presence [vpr(F34I)-off] or in the absence [vpr(F34I)-on] of thiamine to induce vpr gene expression. Cell length was measured as described for Fig. 1A. Immunoblot analysis was performed to confirm the expression of vpr in the srk1⁺ and Δsrk1 strains. The vpr-repressing and vpr-expressing cells were collected and lysed after 20 h of gene induction. Equal amounts of proteins (25 μg) were loaded onto SDS-PAGE gels, and the Vpr was visualized by using anti-Vpr (dilution, 1/500). The same levels of Vpr proteins were expressed in each of these samples (data not shown). (C) Vpr directly interacts with Srk1, as indicated by immunoprecipitation. The RE007 yeast strain was transformed with pREP1-srk1(6HisHA), pREP1-srk1(K153A)(6HisHA), or pREP1-L3(6HisHA) and was grown in the selective liquid culture for 24 h in the presence or in the absence of thiamine to induce gene expression. Srk1 was then immunoprecipitated with anti-HA antibody from cell extracts. The protein L3 was used here as a nonspecific protein control during the immunoprecipitation experiment. G.I., gene induction; IP, immunoprecipitation; TE, total extract. Bar, 10 μm.

Putative model for Srk1/MK2-mediated nuclear-cytoplasmic shuttling of Cdc25 during cell cycle G₂ arrest induced by Vpr.

Vpr-mediated nuclear exclusion of Cdc25 is Rad24 and Crm1 dependent. (A) Rad24-dependent nuclear exclusion. The RE109 (rad24⁺ vpr), Q2019 (Δrad24), and SH3 (Δrad24 vpr) fission yeast strains were grown in liquid culture for 20 h either in the presence (vpr-off) or in the absence (vpr-on) of thiamine to induce gene expression. Immunoblot analysis was performed to confirm the proper expression of vpr in RE109 and SH3 cells. The same levels of Vpr proteins were expressed in each of these samples (data not shown). (B) Treatment of vpr-expressing cells with the nuclear export Crm1 inhibitor LMB blocks the Vpr-mediated nuclear exclusion of Cdc25. The RE109 fission yeast strain was grown in liquid culture for 20 h either in the presence (vpr-off) or in the absence (vpr-on) of thiamine to induce gene expression. When indicated, LMB was added to the culture at a final concentration of 20 ng/ml as previously described (4, 61), and cellular localization of Cdc25 were performed 1 h after the treatment. Immunoblot analysis was performed to confirm the proper expression of vpr in the RE109 cells either in the presence or in the absence of LMB. For the immunoblot analyses, all cells were collected and lysed 20 h after gene induction. Equal amounts of proteins (25 μg) were loaded onto SDS-PAGE gels, and the Vpr was visualized with anti-Vpr (dilution, 1/500). The same levels of Vpr proteins were expressed in each of these samples (data not shown). Subcellular localization of Cdc25-GFP was visualized microscopically by the emission of green fluorescence. Bar, 10 μm.

Kinase Srk1, instead of Chk1 or Cds1, is required by Vpr for the nuclear export of Cdc25. (A) Deletions of the chk1, cds1, or chk1/cds1 genes are unable to block Vpr-mediated nuclear export of Cdc25, but deletion of the srk1 gene abrogated the Vpr effect on Cdc25. All of the fission yeast strains, i.e., the wild-type (SP223), Δsrk1 (RA112), Δchk1 (SH4), Δchk1/Δcds1 (SH5), and Δcds1 (SH6) strains, were transformed with either pREP41x-gfp-cdc25/pYZ2N-vpr or pREP41x-gfp-cdc25, respectively. Cells were grown for 20 h in minimal EMM selective liquid medium in the absence of thiamine to induce gene expression. Cell images were captured to document the cell morphology, and the localization of Cdc25-GFP was visualized by viewing the emission of green fluorescence with a Leica fluorescence microscope. Immunoblot analysis was performed to confirm the proper expression of vpr in every yeast strain. The vpr-repressing and vpr-expressing cells were collected and lysed 20 h after gene induction. Equal amounts of proteins (25 μg) were loaded onto SDS-PAGE gels, and Vpr was visualized with anti-Vpr (dilution, 1/500). The same levels of Vpr proteins were expressed in each of these samples (data not shown). (B) Deletion of the srk1 gene has no obvious effect on the localization of Cdc25 under normal growth conditions. The srk1⁺ and Δsrk1 yeast strains were transformed with pREP41x-GFP, pREP41x-GFP-cdc25, or pREP41x-GFP-cdc25(9A) and grown and assayed as described for panel A. Bar, 10 μm.

MK2, a human homologue of Srk1, is required for Vpr-induced cell cycle G₂ arrest in mammalian cells. (A) Treatment of vpr-expressing HEK293 cells with the MK2 inhibitor (MK2a) abolished Vpr-induced G₂ arrest. (a) Cell cycle profile analysis by flow cytometry. The DL-3 cells, which are derivatives of HEK293 carrying a stable and muristerone A-inducible vpr gene (34, 67), were grown in Dulbecco modified Eagle medium, and the expression of vpr was induced by the addition of muristerone A at a final concentration of 1 μM. At 24 h prior to vpr gene induction, the MK2-specific inhibitor (MK2a) was added at a final concentration of 50 μM. The cells were collected 72 h after gene induction for flow cytometry analysis. (b) Immunoblotting was performed to confirm the expression of vpr in DL-3 cells. The vpr-repressing and vpr-expressing cells were prepared as described for the flow cytometric analysis. Cells were collected and lysed 72 h after gene induction. Equal amounts of proteins (50 μg) were loaded onto SDS-PAGE gels, and the Vpr was visualized by using anti-Vpr (dilution, 1/300). (B) Depletion of MK2 gene expression by siRNA abrogated Vpr-induced G₂ arrest. (a) The DL-3 cells were prepared as described for panel A. At 24 h prior to vpr gene induction, siRNA-MK2 was transfected into the cells. The cells were collected 72 h after gene induction for flow cytometry analysis. Reduction of the MK2 protein production was confirmed by the immunoblot analysis shown in (shown in panel Bb). (c) Images of HEK293 cell morphology with (+) or without (−) the knockdown of MK2 were captured by Leica microscopy with cells collected 72 h after the addition of siRNA-MK2. (C) Depletion of MK2 reduces Cdc25C-Ser216 phosphorylation and partially restores the Cdc25C protein level in the presence of Vpr. The DL-3 cells described for panel Bc with or without the knockdown of MK2 were collected at the indicated times. The proteins extracted were subjected to the Western blot analysis. Phosphorylation of Cdc25C at the Ser216 residue was detected by using monoclonal anti-Cdc25C antibody (Cell Signal). Total protein levels of Cdc25C and MK2 were detected by using a monoclonal antibody (UpState) and polyclonal anti-MK2 antibody (Cell Signaling), respectively.