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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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hCdc14B dephosphorylates Cdc25.A. hCdc14B dephosphorylates Cdc25 in vivo. Immunoblots of nocodazole-arrested HEK293T cells co-expressing fl-hCdc14B or fl-hCdc14BSL and HA-Cdc25A, HA-Cdc25B or GFP-Cdc25C. The upper bands correspond to hyperphosphorylated Cdc25 proteins. B. Cdc25 phosphatases are hyperphosphorylated in cells depleted of hCdc14B. HeLa Kyoto cells transfected with control (siCtrl) or hCdc14B-specific siRNA-3 (siCdc14B-3) were released from G1/S for 18 h, and analyzed on Western blots. C. hCdc14B interacts with Cdc25 isoforms in vivo. Immunoblots showing co-precipitation of Cdc25 isoforms with fl-hCdc14BSL from 293T cells co-expressing HA-Cdc25A, HA-Cdc25B, GFP-Cdc25C, and fl-hCdc14BSL. D. Overexpression of hCdc14B leads to inactivation of Cdc25A. Cdc25A was immunoprecipitated from doxycyclin-treated (dox) or untreated U2OS-fl-hCdc14B cells, incubated with Cdk1/cyclin B from S-phase cells, and kinase activity of Cdk1/cyclin B was monitored by in vitro phosphorylation of histone H1 (top). As a control, activation of Cdk1/cyclin B was monitored using recombinant GST-Cdc25A (bottom). Quantification of histone H1 phosphorylation was performed with a PhosphorImager and is shown below. E. Model depicting the role of hCdc14B during mitosis. Entry into mitosis requires a positive feedback loop, involving Cdk1-dependent activation of Cdc25 phosphatases and Cdc25-dependent dephosphorylation of Cdk1 on pT14/Y15. Before onset of mitosis, hCdc14B is sequestered within the nucleolus, preventing premature dephosphorylation and inactivation of Cdc25 proteins. From prometaphase until late mitosis, hCdc14B is released from nucleolar chromatin, leading to inactivation of Cdc25s, which triggers inhibitory phosphorylation of Cdk1 and inactivation of Cdk1/cyclin B.
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pone-0014711-g006: hCdc14B dephosphorylates Cdc25.A. hCdc14B dephosphorylates Cdc25 in vivo. Immunoblots of nocodazole-arrested HEK293T cells co-expressing fl-hCdc14B or fl-hCdc14BSL and HA-Cdc25A, HA-Cdc25B or GFP-Cdc25C. The upper bands correspond to hyperphosphorylated Cdc25 proteins. B. Cdc25 phosphatases are hyperphosphorylated in cells depleted of hCdc14B. HeLa Kyoto cells transfected with control (siCtrl) or hCdc14B-specific siRNA-3 (siCdc14B-3) were released from G1/S for 18 h, and analyzed on Western blots. C. hCdc14B interacts with Cdc25 isoforms in vivo. Immunoblots showing co-precipitation of Cdc25 isoforms with fl-hCdc14BSL from 293T cells co-expressing HA-Cdc25A, HA-Cdc25B, GFP-Cdc25C, and fl-hCdc14BSL. D. Overexpression of hCdc14B leads to inactivation of Cdc25A. Cdc25A was immunoprecipitated from doxycyclin-treated (dox) or untreated U2OS-fl-hCdc14B cells, incubated with Cdk1/cyclin B from S-phase cells, and kinase activity of Cdk1/cyclin B was monitored by in vitro phosphorylation of histone H1 (top). As a control, activation of Cdk1/cyclin B was monitored using recombinant GST-Cdc25A (bottom). Quantification of histone H1 phosphorylation was performed with a PhosphorImager and is shown below. E. Model depicting the role of hCdc14B during mitosis. Entry into mitosis requires a positive feedback loop, involving Cdk1-dependent activation of Cdc25 phosphatases and Cdc25-dependent dephosphorylation of Cdk1 on pT14/Y15. Before onset of mitosis, hCdc14B is sequestered within the nucleolus, preventing premature dephosphorylation and inactivation of Cdc25 proteins. From prometaphase until late mitosis, hCdc14B is released from nucleolar chromatin, leading to inactivation of Cdc25s, which triggers inhibitory phosphorylation of Cdk1 and inactivation of Cdk1/cyclin B.

Mentions: Phosphorylation of Cdc25 promotes activation of Cdk1, inactivation of Cdc25 correlating with repression of Cdk1 activity [35]. We therefore investigated whether hCdc14B targets Cdc25 phosphatases, dephosphorylation of Cdc25 proteins by hCdc14B promoting inactivation of Cdk1 and completion of mitosis. Indeed, wildtype but not mutant hCdc14B or alkaline phosphatase (CIAP) efficiently dephosphorylated all three isoforms of Cdc25 in vitro (Supp. Fig. S5A and B). To prove that hCdc14B also targeted Cdc25s in vivo, cells co-expressing fl-hCdc14B and the different isoforms of Cdc25 were arrested in mitosis, and phosphorylation of Cdc25A, B and C was analyzed on immunoblots. Consistent with the in vitro data, fl-hCdc14B dephosphorylated all three isoforms of Cdc25, shifting the hyperphosphorylated to the faster migrating hypophosphorylated form (Fig. 6A). Notably, siRNA-mediated depletion of hCdc14B increased the level of hyperphosphorylated, active Cdc25A, B and C (Fig. 6B) demonstrating a link between hCdc14B and the phosphorylation level of all three Cdc25 isoforms.


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)

hCdc14B dephosphorylates Cdc25.A. hCdc14B dephosphorylates Cdc25 in vivo. Immunoblots of nocodazole-arrested HEK293T cells co-expressing fl-hCdc14B or fl-hCdc14BSL and HA-Cdc25A, HA-Cdc25B or GFP-Cdc25C. The upper bands correspond to hyperphosphorylated Cdc25 proteins. B. Cdc25 phosphatases are hyperphosphorylated in cells depleted of hCdc14B. HeLa Kyoto cells transfected with control (siCtrl) or hCdc14B-specific siRNA-3 (siCdc14B-3) were released from G1/S for 18 h, and analyzed on Western blots. C. hCdc14B interacts with Cdc25 isoforms in vivo. Immunoblots showing co-precipitation of Cdc25 isoforms with fl-hCdc14BSL from 293T cells co-expressing HA-Cdc25A, HA-Cdc25B, GFP-Cdc25C, and fl-hCdc14BSL. D. Overexpression of hCdc14B leads to inactivation of Cdc25A. Cdc25A was immunoprecipitated from doxycyclin-treated (dox) or untreated U2OS-fl-hCdc14B cells, incubated with Cdk1/cyclin B from S-phase cells, and kinase activity of Cdk1/cyclin B was monitored by in vitro phosphorylation of histone H1 (top). As a control, activation of Cdk1/cyclin B was monitored using recombinant GST-Cdc25A (bottom). Quantification of histone H1 phosphorylation was performed with a PhosphorImager and is shown below. E. Model depicting the role of hCdc14B during mitosis. Entry into mitosis requires a positive feedback loop, involving Cdk1-dependent activation of Cdc25 phosphatases and Cdc25-dependent dephosphorylation of Cdk1 on pT14/Y15. Before onset of mitosis, hCdc14B is sequestered within the nucleolus, preventing premature dephosphorylation and inactivation of Cdc25 proteins. From prometaphase until late mitosis, hCdc14B is released from nucleolar chromatin, leading to inactivation of Cdc25s, which triggers inhibitory phosphorylation of Cdk1 and inactivation of Cdk1/cyclin B.
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Related In: Results  -  Collection

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pone-0014711-g006: hCdc14B dephosphorylates Cdc25.A. hCdc14B dephosphorylates Cdc25 in vivo. Immunoblots of nocodazole-arrested HEK293T cells co-expressing fl-hCdc14B or fl-hCdc14BSL and HA-Cdc25A, HA-Cdc25B or GFP-Cdc25C. The upper bands correspond to hyperphosphorylated Cdc25 proteins. B. Cdc25 phosphatases are hyperphosphorylated in cells depleted of hCdc14B. HeLa Kyoto cells transfected with control (siCtrl) or hCdc14B-specific siRNA-3 (siCdc14B-3) were released from G1/S for 18 h, and analyzed on Western blots. C. hCdc14B interacts with Cdc25 isoforms in vivo. Immunoblots showing co-precipitation of Cdc25 isoforms with fl-hCdc14BSL from 293T cells co-expressing HA-Cdc25A, HA-Cdc25B, GFP-Cdc25C, and fl-hCdc14BSL. D. Overexpression of hCdc14B leads to inactivation of Cdc25A. Cdc25A was immunoprecipitated from doxycyclin-treated (dox) or untreated U2OS-fl-hCdc14B cells, incubated with Cdk1/cyclin B from S-phase cells, and kinase activity of Cdk1/cyclin B was monitored by in vitro phosphorylation of histone H1 (top). As a control, activation of Cdk1/cyclin B was monitored using recombinant GST-Cdc25A (bottom). Quantification of histone H1 phosphorylation was performed with a PhosphorImager and is shown below. E. Model depicting the role of hCdc14B during mitosis. Entry into mitosis requires a positive feedback loop, involving Cdk1-dependent activation of Cdc25 phosphatases and Cdc25-dependent dephosphorylation of Cdk1 on pT14/Y15. Before onset of mitosis, hCdc14B is sequestered within the nucleolus, preventing premature dephosphorylation and inactivation of Cdc25 proteins. From prometaphase until late mitosis, hCdc14B is released from nucleolar chromatin, leading to inactivation of Cdc25s, which triggers inhibitory phosphorylation of Cdk1 and inactivation of Cdk1/cyclin B.
Mentions: Phosphorylation of Cdc25 promotes activation of Cdk1, inactivation of Cdc25 correlating with repression of Cdk1 activity [35]. We therefore investigated whether hCdc14B targets Cdc25 phosphatases, dephosphorylation of Cdc25 proteins by hCdc14B promoting inactivation of Cdk1 and completion of mitosis. Indeed, wildtype but not mutant hCdc14B or alkaline phosphatase (CIAP) efficiently dephosphorylated all three isoforms of Cdc25 in vitro (Supp. Fig. S5A and B). To prove that hCdc14B also targeted Cdc25s in vivo, cells co-expressing fl-hCdc14B and the different isoforms of Cdc25 were arrested in mitosis, and phosphorylation of Cdc25A, B and C was analyzed on immunoblots. Consistent with the in vitro data, fl-hCdc14B dephosphorylated all three isoforms of Cdc25, shifting the hyperphosphorylated to the faster migrating hypophosphorylated form (Fig. 6A). Notably, siRNA-mediated depletion of hCdc14B increased the level of hyperphosphorylated, active Cdc25A, B and C (Fig. 6B) demonstrating a link between hCdc14B and the phosphorylation level of all three Cdc25 isoforms.

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