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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 regulates Cdk1/cyclin B activity.A. Overexpression of hCdc14B alters Cdk1/cyclin B activity. Cdk1/cyclin B was immunopurified from U2OS, U2OS-fl-hCdc14B and U2OS-fl-hCdc14BSL cells that were synchronized at G1/S (0 h) and released for the indicated times. Kinase activity was assayed in vitro using Cdk1-specific peptides and 32P-ATP. Values were normalized to the activity of Cdk1/cyclin B in G1/S cells. The Western blots show the amount of immunoprecipitated cyclin B, Cdk1 and Cdk1/pY15. B. Depletion of hCdc14B increases Cdk1/cyclin B activity. Cells were transfected with GFP- (siGFP, light bars) or hCdc14B-specific siRNA-2 (siCdc14B-2, dark bars) and arrested by double thymidine block. Kinase activity of immunopurified Cdk1/cyclin B was assayed in vitro at the indicated times (h) after release from G1/S. The immunoblot on the top shows the amounts of cyclin B used in the assays. C. Cyclin B is stabilized upon knockdown of hCdc14B. Cells were transfected with siRNAs against GFP (siGFP), hCdc14B (siCdc14B-2 and siCdc14B-3) or left untransfected (mock), synchronized by double thymidine block, released from G1/S for 16 h and analyzed on immunoblots.
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pone-0014711-g005: hCdc14B regulates Cdk1/cyclin B activity.A. Overexpression of hCdc14B alters Cdk1/cyclin B activity. Cdk1/cyclin B was immunopurified from U2OS, U2OS-fl-hCdc14B and U2OS-fl-hCdc14BSL cells that were synchronized at G1/S (0 h) and released for the indicated times. Kinase activity was assayed in vitro using Cdk1-specific peptides and 32P-ATP. Values were normalized to the activity of Cdk1/cyclin B in G1/S cells. The Western blots show the amount of immunoprecipitated cyclin B, Cdk1 and Cdk1/pY15. B. Depletion of hCdc14B increases Cdk1/cyclin B activity. Cells were transfected with GFP- (siGFP, light bars) or hCdc14B-specific siRNA-2 (siCdc14B-2, dark bars) and arrested by double thymidine block. Kinase activity of immunopurified Cdk1/cyclin B was assayed in vitro at the indicated times (h) after release from G1/S. The immunoblot on the top shows the amounts of cyclin B used in the assays. C. Cyclin B is stabilized upon knockdown of hCdc14B. Cells were transfected with siRNAs against GFP (siGFP), hCdc14B (siCdc14B-2 and siCdc14B-3) or left untransfected (mock), synchronized by double thymidine block, released from G1/S for 16 h and analyzed on immunoblots.

Mentions: Reversal of mitotic Cdk1-dependent phosphorylations and inhibition of Cdk1 are hallmarks of Cdc14 function in yeast [18], [34]. To decipher a functional link between human Cdc14B and Cdk1/cyclin B, we examined whether changes in hCdc14B levels would affect Cdk1 activity. For this, we compared activation of Cdk1/cyclin B in U2OS cell lines conditionally expressing fl-hCdc14B or fl-hCdc14BSL. After induction of wildtype or mutant fl-hCdc14B in G1/S arrested cells (0 h), kinase activity of immunopurified Cdk1/cyclin B was assayed at distinct times after release. In parental cells, Cdk1/cyclin B activity reached maximal levels after 12–13 h, and then declined because of cyclin B degradation (Fig. 5A, left). In contrast and consistent with data shown in Figure 1B, activation of Cdk1 was delayed in cells expressing fl-hCdc14B, reaching maximal levels at 15 h, which declined more slowly than in parental cells (Fig. 5A, middle). The delay in Cdk1 activation correlated with elevated levels of pY15 (Fig. 5A, middle). Cdk1 activity was high over an extended period in cells expressing the catalytically inactive mutant fl-hCdc14BSL (Fig. 5A, right), suggesting that hCdc14B phosphatase is required for timely inactivation of Cdk1/cyclin B.


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 regulates Cdk1/cyclin B activity.A. Overexpression of hCdc14B alters Cdk1/cyclin B activity. Cdk1/cyclin B was immunopurified from U2OS, U2OS-fl-hCdc14B and U2OS-fl-hCdc14BSL cells that were synchronized at G1/S (0 h) and released for the indicated times. Kinase activity was assayed in vitro using Cdk1-specific peptides and 32P-ATP. Values were normalized to the activity of Cdk1/cyclin B in G1/S cells. The Western blots show the amount of immunoprecipitated cyclin B, Cdk1 and Cdk1/pY15. B. Depletion of hCdc14B increases Cdk1/cyclin B activity. Cells were transfected with GFP- (siGFP, light bars) or hCdc14B-specific siRNA-2 (siCdc14B-2, dark bars) and arrested by double thymidine block. Kinase activity of immunopurified Cdk1/cyclin B was assayed in vitro at the indicated times (h) after release from G1/S. The immunoblot on the top shows the amounts of cyclin B used in the assays. C. Cyclin B is stabilized upon knockdown of hCdc14B. Cells were transfected with siRNAs against GFP (siGFP), hCdc14B (siCdc14B-2 and siCdc14B-3) or left untransfected (mock), synchronized by double thymidine block, released from G1/S for 16 h and analyzed on immunoblots.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040744&req=5

pone-0014711-g005: hCdc14B regulates Cdk1/cyclin B activity.A. Overexpression of hCdc14B alters Cdk1/cyclin B activity. Cdk1/cyclin B was immunopurified from U2OS, U2OS-fl-hCdc14B and U2OS-fl-hCdc14BSL cells that were synchronized at G1/S (0 h) and released for the indicated times. Kinase activity was assayed in vitro using Cdk1-specific peptides and 32P-ATP. Values were normalized to the activity of Cdk1/cyclin B in G1/S cells. The Western blots show the amount of immunoprecipitated cyclin B, Cdk1 and Cdk1/pY15. B. Depletion of hCdc14B increases Cdk1/cyclin B activity. Cells were transfected with GFP- (siGFP, light bars) or hCdc14B-specific siRNA-2 (siCdc14B-2, dark bars) and arrested by double thymidine block. Kinase activity of immunopurified Cdk1/cyclin B was assayed in vitro at the indicated times (h) after release from G1/S. The immunoblot on the top shows the amounts of cyclin B used in the assays. C. Cyclin B is stabilized upon knockdown of hCdc14B. Cells were transfected with siRNAs against GFP (siGFP), hCdc14B (siCdc14B-2 and siCdc14B-3) or left untransfected (mock), synchronized by double thymidine block, released from G1/S for 16 h and analyzed on immunoblots.
Mentions: Reversal of mitotic Cdk1-dependent phosphorylations and inhibition of Cdk1 are hallmarks of Cdc14 function in yeast [18], [34]. To decipher a functional link between human Cdc14B and Cdk1/cyclin B, we examined whether changes in hCdc14B levels would affect Cdk1 activity. For this, we compared activation of Cdk1/cyclin B in U2OS cell lines conditionally expressing fl-hCdc14B or fl-hCdc14BSL. After induction of wildtype or mutant fl-hCdc14B in G1/S arrested cells (0 h), kinase activity of immunopurified Cdk1/cyclin B was assayed at distinct times after release. In parental cells, Cdk1/cyclin B activity reached maximal levels after 12–13 h, and then declined because of cyclin B degradation (Fig. 5A, left). In contrast and consistent with data shown in Figure 1B, activation of Cdk1 was delayed in cells expressing fl-hCdc14B, reaching maximal levels at 15 h, which declined more slowly than in parental cells (Fig. 5A, middle). The delay in Cdk1 activation correlated with elevated levels of pY15 (Fig. 5A, middle). Cdk1 activity was high over an extended period in cells expressing the catalytically inactive mutant fl-hCdc14BSL (Fig. 5A, right), suggesting that hCdc14B phosphatase is required for timely inactivation of Cdk1/cyclin B.

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