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Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity.

Tumurbaatar I, Cizmecioglu O, Hoffmann I, Grummt I, Voit R - PLoS ONE (2011)

Bottom Line: Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known.Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis.Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology of the Cell II, German Cancer Research Centre, DKFZ-ZMBH Alliance, Heidelberg, Germany.

ABSTRACT
Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

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Depletion of hCdc14B impairs progression through mitosis and triggers apoptosis.A. Cell synchronization and specificity of hCdc14B knockdown by siRNAs. HeLa Kyoto cells were reverse transfected with a control siRNA pool (siCtrl), siRNAs that target GFP-mRNA (siGFP), or three different regions of hCdc14B-mRNA (siCdc14B-1, -2, -3; see Table S1 for sequence information) and synchronized as shown in the scheme. 8 h after release from the second G1/S arrest, cells were lysed and the amount of hCdc14B, hCdc14A, H2B-GFP, and actin was analyzed on immunoblots. B. Phase contrast and H2B-GFP fluorescence images of HeLa Kyoto cells that were transfected with control-siRNA (siCtrl) or hCdc14B-siRNA (siCdc14B-3) and released from G1/S according to the scheme above. Before the second G1/S arrest, equal numbers of cells were seeded onto coverslips. The GFP images represent magnifications of regions marked by rectangles in the phase contrast images. C. Western blot analysis of Cdc27/APC3, hCdc14B, and β-tubulin in lysates from cells treated with GFP-specific (siGFP) or hCdc14B-specific (siCdc14B-3) siRNAs. The hyperphosphorylated form of Cdc27/APC3 is indicated. D. hCdc14B depletion induces apoptosis. siRNA transfections and cell synchronization was done as described above. Before the second thymidine block, equal numbers of cells were seeded on coverslips and apoptosis was monitored 24 h after release from the second G1/S-arrest in control (siGFP) or hCdc14B-depleted (siCdc14B-3) HeLa Kyoto cells by TUNEL staining (left) and on immunoblots monitoring caspase-induced proteolysis of PARP-1 (right).
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pone-0014711-g003: Depletion of hCdc14B impairs progression through mitosis and triggers apoptosis.A. Cell synchronization and specificity of hCdc14B knockdown by siRNAs. HeLa Kyoto cells were reverse transfected with a control siRNA pool (siCtrl), siRNAs that target GFP-mRNA (siGFP), or three different regions of hCdc14B-mRNA (siCdc14B-1, -2, -3; see Table S1 for sequence information) and synchronized as shown in the scheme. 8 h after release from the second G1/S arrest, cells were lysed and the amount of hCdc14B, hCdc14A, H2B-GFP, and actin was analyzed on immunoblots. B. Phase contrast and H2B-GFP fluorescence images of HeLa Kyoto cells that were transfected with control-siRNA (siCtrl) or hCdc14B-siRNA (siCdc14B-3) and released from G1/S according to the scheme above. Before the second G1/S arrest, equal numbers of cells were seeded onto coverslips. The GFP images represent magnifications of regions marked by rectangles in the phase contrast images. C. Western blot analysis of Cdc27/APC3, hCdc14B, and β-tubulin in lysates from cells treated with GFP-specific (siGFP) or hCdc14B-specific (siCdc14B-3) siRNAs. The hyperphosphorylated form of Cdc27/APC3 is indicated. D. hCdc14B depletion induces apoptosis. siRNA transfections and cell synchronization was done as described above. Before the second thymidine block, equal numbers of cells were seeded on coverslips and apoptosis was monitored 24 h after release from the second G1/S-arrest in control (siGFP) or hCdc14B-depleted (siCdc14B-3) HeLa Kyoto cells by TUNEL staining (left) and on immunoblots monitoring caspase-induced proteolysis of PARP-1 (right).

Mentions: We next examined mitotic progression upon depletion of hCdc14B in synchronized HeLa Kyoto cells (Fig. 3A). After release from the second G1/S-arrest, progression into mitosis was monitored by live microscopy visualizing chromosome condensation via H2B-GFP-labeled chromatin. Representative images of the phenotypic responses from RNAi with an hCdc14B-specific siRNA are shown in Figure 3B. Cells entered mitosis 12 h after release, regardless whether or not hCdc14B was depleted. While most of control cells re-entered a new division cycle at 16 h after release from G1/S, a significant proportion (48%) of hCdc14B-depleted cells was enriched in metaphase (Fig. 3B and Table 1), suggesting that hCdc14B activity is required for successful completion of mitosis. Consistent with the delay of metaphase to anaphase transition, prolonged mitotic hyperphosphorylation of Cdc27/APC3 [33] was observed (Fig. 3C). Moreover, 24 h after release a large number of hCdc14B-siRNA treated cells stained positive in the TUNEL assay with cleavage of PARP-1 (Fig. 3D and Table 1), indicating that the strong delay in mid-/late-mitosis has triggered apoptosis. Again and consistent with knockdown of hCdc14B in non-synchronized cells (Fig. 2), the amount of bi- and multinucleated cells was increased (Supp. Fig. S3 and Table 1). Together, these results strongly suggest that downregulation of hCdc14B impairs metaphase-anaphase transition mitotic delay leading to cell death.


Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity.

Tumurbaatar I, Cizmecioglu O, Hoffmann I, Grummt I, Voit R - PLoS ONE (2011)

Depletion of hCdc14B impairs progression through mitosis and triggers apoptosis.A. Cell synchronization and specificity of hCdc14B knockdown by siRNAs. HeLa Kyoto cells were reverse transfected with a control siRNA pool (siCtrl), siRNAs that target GFP-mRNA (siGFP), or three different regions of hCdc14B-mRNA (siCdc14B-1, -2, -3; see Table S1 for sequence information) and synchronized as shown in the scheme. 8 h after release from the second G1/S arrest, cells were lysed and the amount of hCdc14B, hCdc14A, H2B-GFP, and actin was analyzed on immunoblots. B. Phase contrast and H2B-GFP fluorescence images of HeLa Kyoto cells that were transfected with control-siRNA (siCtrl) or hCdc14B-siRNA (siCdc14B-3) and released from G1/S according to the scheme above. Before the second G1/S arrest, equal numbers of cells were seeded onto coverslips. The GFP images represent magnifications of regions marked by rectangles in the phase contrast images. C. Western blot analysis of Cdc27/APC3, hCdc14B, and β-tubulin in lysates from cells treated with GFP-specific (siGFP) or hCdc14B-specific (siCdc14B-3) siRNAs. The hyperphosphorylated form of Cdc27/APC3 is indicated. D. hCdc14B depletion induces apoptosis. siRNA transfections and cell synchronization was done as described above. Before the second thymidine block, equal numbers of cells were seeded on coverslips and apoptosis was monitored 24 h after release from the second G1/S-arrest in control (siGFP) or hCdc14B-depleted (siCdc14B-3) HeLa Kyoto cells by TUNEL staining (left) and on immunoblots monitoring caspase-induced proteolysis of PARP-1 (right).
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Related In: Results  -  Collection

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pone-0014711-g003: Depletion of hCdc14B impairs progression through mitosis and triggers apoptosis.A. Cell synchronization and specificity of hCdc14B knockdown by siRNAs. HeLa Kyoto cells were reverse transfected with a control siRNA pool (siCtrl), siRNAs that target GFP-mRNA (siGFP), or three different regions of hCdc14B-mRNA (siCdc14B-1, -2, -3; see Table S1 for sequence information) and synchronized as shown in the scheme. 8 h after release from the second G1/S arrest, cells were lysed and the amount of hCdc14B, hCdc14A, H2B-GFP, and actin was analyzed on immunoblots. B. Phase contrast and H2B-GFP fluorescence images of HeLa Kyoto cells that were transfected with control-siRNA (siCtrl) or hCdc14B-siRNA (siCdc14B-3) and released from G1/S according to the scheme above. Before the second G1/S arrest, equal numbers of cells were seeded onto coverslips. The GFP images represent magnifications of regions marked by rectangles in the phase contrast images. C. Western blot analysis of Cdc27/APC3, hCdc14B, and β-tubulin in lysates from cells treated with GFP-specific (siGFP) or hCdc14B-specific (siCdc14B-3) siRNAs. The hyperphosphorylated form of Cdc27/APC3 is indicated. D. hCdc14B depletion induces apoptosis. siRNA transfections and cell synchronization was done as described above. Before the second thymidine block, equal numbers of cells were seeded on coverslips and apoptosis was monitored 24 h after release from the second G1/S-arrest in control (siGFP) or hCdc14B-depleted (siCdc14B-3) HeLa Kyoto cells by TUNEL staining (left) and on immunoblots monitoring caspase-induced proteolysis of PARP-1 (right).
Mentions: We next examined mitotic progression upon depletion of hCdc14B in synchronized HeLa Kyoto cells (Fig. 3A). After release from the second G1/S-arrest, progression into mitosis was monitored by live microscopy visualizing chromosome condensation via H2B-GFP-labeled chromatin. Representative images of the phenotypic responses from RNAi with an hCdc14B-specific siRNA are shown in Figure 3B. Cells entered mitosis 12 h after release, regardless whether or not hCdc14B was depleted. While most of control cells re-entered a new division cycle at 16 h after release from G1/S, a significant proportion (48%) of hCdc14B-depleted cells was enriched in metaphase (Fig. 3B and Table 1), suggesting that hCdc14B activity is required for successful completion of mitosis. Consistent with the delay of metaphase to anaphase transition, prolonged mitotic hyperphosphorylation of Cdc27/APC3 [33] was observed (Fig. 3C). Moreover, 24 h after release a large number of hCdc14B-siRNA treated cells stained positive in the TUNEL assay with cleavage of PARP-1 (Fig. 3D and Table 1), indicating that the strong delay in mid-/late-mitosis has triggered apoptosis. Again and consistent with knockdown of hCdc14B in non-synchronized cells (Fig. 2), the amount of bi- and multinucleated cells was increased (Supp. Fig. S3 and Table 1). Together, these results strongly suggest that downregulation of hCdc14B impairs metaphase-anaphase transition mitotic delay leading to cell death.

Bottom Line: Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known.Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis.Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology of the Cell II, German Cancer Research Centre, DKFZ-ZMBH Alliance, Heidelberg, Germany.

ABSTRACT
Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

Show MeSH
Related in: MedlinePlus