Cdk1-Mediated Phosphorylation of Human ATF7 at Thr-51 and Thr-53 Promotes Cell-Cycle Progression into M Phase

<div><p>Activating transcription factor 2 (ATF2) and its homolog ATF7 are phosphorylated at Thr-69/Thr-71 and at Thr-51/Thr-53, respectively, by stress-activated MAPKs regulating their transcriptional functions in G1 and S phases. However, little is known about the role of ATF2 and ATF7 in G2/M phase. Here, we show that Cdk1-cyclin B1 phosphorylates ATF2 at Thr-69/Thr-71 and ATF7 at Thr-51/Thr-53 from early prophase to anaphase in the absence of any stress stimulation. Knockdown of ATF2 or ATF7 decreases the rate of cell proliferation and the number of cells in M-phase. In particular, the knockdown of ATF7 severely inhibits cell proliferation and G2/M progression. The inducible expression of a mitotically nonphosphorylatable version of ATF7 inhibits G2/M progression despite the presence of endogenous ATF7. We also show that mitotic phosphorylation of ATF7 promotes the activation of Aurora kinases, which are key enzymes for early mitotic events. These results suggest that the Cdk1-mediated phosphorylation of ATF7 facilitates G2/M progression, at least in part, by enabling Aurora signaling.</p></div

Differential roles for the phosphorylation of ATF7 and ATF2 in G1/S phases and G2/M phases.

<p>In G1 and S phases, ATF7 and ATF2 are phosphorylated at Thr-51 and Thr-53 and at Thr-69 and Thr-71, respectively, by stress-activated MAP kinases, to induce their transcriptional functions. During late G2 and M phases, the Thr-51/Thr-53 on ATF7 and the Thr-69/Thr-71 on ATF2 are phosphorylated by Cdk1-cyclin B1, which promotes M-phase entry via the stabilization of Aurora kinases.</p

Mitotic phosphorylation of ATF2 and ATF7 by Cdk1.

<p>(<b>A</b>) Cells were synchronized at S, G2, or M phases. Mitotic cells were collected by mitotic shake-off, and whole cell lysates were analyzed by WB using antibodies against ATF2/ATF7 (ATF2[N96]), pATF2/pATF7 (pT69/71), cyclin B1, and actin. (<b>B–D</b> Cells were arrested at G2 phase using 9 µM RO-3306 and released into RO-3306-free medium containing 10 µM MG132. (<b>B</b>) At 20 min after release, cells were treated for an additional 60 min in the presence of 10 µM MG132 together with DMSO (solvent control) or 9 µM RO-3306. Cells were doubly stained with anti-pT69/71 antibody and PI. Cells stained with anti-histone pT69/71 antibody are pseudo-colored as green. Scale bars, 20 µm. (<b>C</b>) At 20 min after release from RO-3306, cells were treated with DMSO (solvent control), 9 µM RO-3306, 1 µM MLN8237, 10 µM ZM447439, 1 µM BI2536, 20 µM SB202190, 20 µM SP600125, or 20 µM U0126 for a further 30 min in the presence of 10 µM MG132. Whole cell lysates were analyzed by WB. Asyn, asynchronous. (<b>D</b>) At 20 min after release, cells were treated for an additional 30 or 60 min in the presence of 10 µM MG132 together with DMSO (solvent control), 9 µM RO-3306, 10 µM Gö6976, or RO-3306 plus Gö6976. Whole cell lysates were analyzed by WB. Asyn, asynchronous. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s007" target="_blank">S7B</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s008" target="_blank">S8A–B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s009" target="_blank">S9 Figs</a>.</p

Phosphorylation of ATF2 (at Thr69/Thr71) and ATF7 (at Thr51/Thr53) by Cdk1-cyclin B1.

<p>(<b>A</b>) Amino acid sequence of ATF2 and ATF7 with the common phosphorylation sites. pATF2, phosphorylated ATF2; pATF7, phosphorylated ATF7; ATF2-TA and ATF7-TA, T→A mutants. (<b>B–E</b> Cells that were transiently transfected with the indicated constructs were cultured for 24 h, and whole cell lysates were analyzed by WB. (<b>B</b>) Cells were cotransfected with ATF2-wt together with control vector, cyclin B1, Cdk1 or cyclin B1 plus Cdk1. (<b>C</b>) Cells were cotransfected with ATF7-wt together with control vector, cyclin B1, Cdk1, or cyclin B1 plus Cdk1. (<b>D</b>) Cells were cotransfected with control vector together with GFP-ATF2-wt or GFP-ATF2-TA, or cells were cotransfected with cyclin B1 plus Cdk1 together with GFP-ATF2-wt or GFP-ATF2-TA. (<b>E</b>) Cells were cotransfected with control vector together with ATF7-wt or ATF7-TA, or cells were cotransfected with cyclin B1 plus Cdk1 together with ATF7-wt or ATF7-TA. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s010" target="_blank">S10 Fig</a>.</p

Effect of ATF2 or ATF7 knockdown on cell-cycle progression.

<p>(<b>A</b>) Schematic depiction of our knockdown method. Cells were transfected with the episomal pEBMulti vector for shRNA expression and selected in 600 µg/ml G418. (<b>B</b>) Cells were transfected with control vector, ATF2 shRNA, ATF7 shRNA(3′UTR), or ATF7 shRNA(CDS) and collected on day 5. Whole cell lysates were analyzed by Western blotting (WB) using anti-ATF2[SS-16], anti-ATF7[HPA003384], and anti-actin (loading control) antibodies. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s007" target="_blank">S7A Fig</a>. (<b>C</b>) The number of cells was counted on days 6∼9 after shRNA transfection. Values are means ± standard deviation (SD), n = 3 independent experiments (only shATF7(CDS), n = 2 independent experiments). Asterisks indicate the significant differences (*P<0.05; **P<0.01), as calculated by Student’s <i>t</i>-test. (<b>D</b>) Cells transfected with control vector, ATF2 shRNA, ATF7 shRNA(3′UTR), or ATF7 shRNA(CDS) were collected on day 7 and stained with propidium iodide (PI). SubG1 cells were quantitated by flow cytometry. Values are means ± SD, n  = 3 independent experiments. Asterisks indicate the significant differences (*P<0.05; **P<0.01), as calculated by Student’s <i>t</i>-test. (<b>E</b>) Schematic depiction of our synchronization method. Cells transfected with control vector, ATF2 shRNA, ATF7 shRNA(3′UTR), or ATF7 shRNA(CDS) were synchronized using double thymidine block (DTB) in the presence of 600 µg/ml G418 and released into thymidine-free medium for 10∼12 h. Cells were stained with PI for analyzing cell cycle progression by flow cytometry (left panels). The percentages of G1-phase cells were compared between 10 h and 12 h after release from DTB (right graph).</p

Involvement of ATF7 phosphorylation in Aurora signaling.

<p>(<b>A</b>) Schematic depiction of our knockdown-rescue experiments. Parental HeLa S3/TR, HeLa S3/TR/ATF7-wt (cl. 2), or HeLa S3/TR/ATF7-TA (cl.1) cells were treated with 1 µg/ml Dox for 12 h and then transfected with shRNAs. Knockdown cells selected using 600 µg/ml G418 in the presence of 1 µg/ml Dox were synchronized by (i) DTB or (ii) thymidine→RO-3306. (<b>B–D</b>) Knockdown cells were synchronized as described in (a)-(i) and collected 12 h after release from DTB. (<b>B</b>) Whole cell lysates were analyzed by WB. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s012" target="_blank">S12B Fig</a>. (<b>C</b>) Cells were stained with PI for analyzing cell-cycle progression by flow cytometry (panels) and for quantitating G1-phase cells (graph). Values are means ± SD, n  = 4 independent experiments. Asterisks indicate the significant differences (*P<0.05; ***P<0.001), as calculated by Student’s <i>t</i>-test. (<b>D, E</b>) Knockdown cells were synchronized as described in (A)-(ii) and collected 40 min after release from RO-3306. (<b>D</b>) Cells were triply stained with anti-ATF7[HPA003384] and anti-histone H3pS10 antibodies (for M phase) and PI (for DNA). Cells stained with anti-histone H3pS10 antibody are pseudo-colored as white. Scale bars, 40 µm. (<b>E</b>) Whole cell lysates were analyzed by WB. *, a nonspecific band. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s013" target="_blank">S13 Fig</a>.</p

Phosphorylation of ATF2 and ATF7 from prophase to anaphase.

<p>(<b>A–E</b> Cells were synchronized using single-thymidine block and released into thymidine-free medium for 11 h. (<b>A</b>) Mitotic cells collected by mitotic shake-off and asynchronous (Asyn) cells were lysed with Triton X-100. Endogenous ATF2, ATF7, and pATF2/pATF7 were individually immunoprecipitated from Triton X-100 cell lysates using antibodies specific for ATF2 [SS-16], ATF7 [SAB2500131], and pATF2/pATF7 (pT69/71). Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s011" target="_blank">S11A Fig</a>. The gels for ATF7 IP blotted with anti-pT69/71 antibody and anti-ATF2 and anti-ATF7 antibodies have been run under the same experimental conditions. (<b>B</b>) Cells were triply stained with anti-pT69/71 and anti-Cdk1 antibodies and PI (for DNA). Anti-Cdk1-stained cells are pseudo-colored as blue. Scale bars, 20 µm. Arrows indicate mitotic cells. (<b>C</b>) Cells were triply stained with anti-pT69/71 and anti-cyclin B1 antibodies and PI (for DNA). Staining of pATF2/pATF7 was recorded with a low sensitivity. Anti-cyclin B1-stained cells are pseudo-colored as blue. Scale bars, 20 µm. (<b>D</b>) Cells were triply stained with anti-ATF2[N96] (upper panels) or anti-pT69/71 (lower panels) antibody, anti-histone H3pS10 antibody (for M phase) and PI (for DNA). Scale bars, 20 µm. (<b>E</b>) Cells were triply stained with anti-pT69/71 antibody, anti-histone H3pS10 antibody (for M phase), and PI (for DNA). Scale bars, 20 µm. (<b>F</b>) Cells were arrested at G2 phase using 9 µM RO-3306 and were released into RO-3306-free medium containing 10 µM MG132. At 20 min after release, cells were treated for an additional 60 min in the presence of 10 µM MG132 together with DMSO, 9 µM RO-3306, 10 µM ZM447439, RO-3306 plus ZM447439, or 20 µM U0126. Whole cell lysates were analyzed by WB. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s011" target="_blank">S11B Fig</a>. (<b>G</b>) Cells arrested at G2 phase by treatment with 9 µM RO-3306 were released into RO-3306-free medium containing 100 µM monastrol for 1 h. The monastrol-arrested cells were collected by mitotic shake off and incubated with 10 µM ZM447439 for the indicated times (induction of mitotic slippage). Whole cell lysates were analyzed by WB. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s011" target="_blank">S11C Fig</a>.</p

Additional file 2: Table S1. of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Molecular and clinical characteristics of Cohort 1 (n = 758). Table S2. Molecular and clinical characteristics of GBM cohort (n = 453). Table S3. Univariate and multivariate Cox regression analyses for Group A (IDH mutated-TERT mutated) tumors in Cohort 1 (n = 155). Table S4. Univariate and multivariate Cox regression analyses for Group B (IDH mutated-TERT wild-type) tumors in Cohort 1 (n = 131). Table S5. Univariate and multivariate Cox regression analyses for Group C (IDH wild-type-TERT wild-type) tumors in Cohort 1 (n = 237). Table S6. Univariate and multivariate Cox regression analyses for Group D (IDH wild-type-TERT mutated) tumors in Cohort 1 (n = 235). Table S7. Univariate and multivariate Cox regression analyses for GBM in Cohort 1 (n = 260). Table S8. Univariate and multivariate Cox regression analyses for GBM in Cohort 2 (n = 193). Table S9. Background of combined GBM cohort stratified by TERT and MGMT status (n = 453). Table S10. Survival time and WHO grade in each molecular subgroup of Cohort 1 (n = 758). (XLSX 254 kb

Additional file 3: Figure S1. of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Distributions of molecular alterations according to histology in Cohort 1. Figure S2. Kaplan-Meier analysis for Group A cases stratified by 1p/19q status. Figure S3. Kaplan-Meier analyses for GBM cases in Cohorts 1 and 2. (PPTX 172 kb

Additional file 1: of A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

Supplementary Information. (DOCX 141 kb