Limits...
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.

Show MeSH

Related in: MedlinePlus

Overexpression of hCdc14B alters progression through mitosis.A. hCdc14B is released from chromatin in early mitosis and is rebound in late M/early G1. HeLa cells expressing histone H2B-GFP (HeLa Kyoto) were arrested at G1/S by double thymidine-block (left) or at prometaphase of mitosis by nocodazole (right), and released for the indicated times. Release from nocodazole (nocod.) arrest was monitored visualizing H2B-GFP labelled chromatin (bottom panel). Synchronized cells were fractionated into cytoplasm (cyt.), nucleoplasm (nucl.) and chromatin (chr.), and analyzed on immunoblots for the proteins indicated (top panels). B. Overexpression of hCdc14B prolongs G2/M-phase. FACS of U2OS, U2OS-fl-hCdc14B and U2OS-fl-hCdc14BSL cells following release from G1/S (0 h) upon induction of hCdc14B expression by doxycyclin (Fig. S1). Cell numbers were calculated using the Cell Quest software program. The percentage of cells at the indicated cell cycle phases is presented. C. Overexpression of hCdc14B affects mitotic progression. Immunoblots showing the abundance of specific cell cycle marker proteins at distinct time points (h) after release from G1/S.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3040744&req=5

pone-0014711-g001: Overexpression of hCdc14B alters progression through mitosis.A. hCdc14B is released from chromatin in early mitosis and is rebound in late M/early G1. HeLa cells expressing histone H2B-GFP (HeLa Kyoto) were arrested at G1/S by double thymidine-block (left) or at prometaphase of mitosis by nocodazole (right), and released for the indicated times. Release from nocodazole (nocod.) arrest was monitored visualizing H2B-GFP labelled chromatin (bottom panel). Synchronized cells were fractionated into cytoplasm (cyt.), nucleoplasm (nucl.) and chromatin (chr.), and analyzed on immunoblots for the proteins indicated (top panels). B. Overexpression of hCdc14B prolongs G2/M-phase. FACS of U2OS, U2OS-fl-hCdc14B and U2OS-fl-hCdc14BSL cells following release from G1/S (0 h) upon induction of hCdc14B expression by doxycyclin (Fig. S1). Cell numbers were calculated using the Cell Quest software program. The percentage of cells at the indicated cell cycle phases is presented. C. Overexpression of hCdc14B affects mitotic progression. Immunoblots showing the abundance of specific cell cycle marker proteins at distinct time points (h) after release from G1/S.

Mentions: Immunofluorescence studies have demonstrated that hCdc14B localizes within nucleoli in interphase cells [23], [28]. Fractionation of extracts from synchronized HeLa Kyoto cells that stably express GFP-tagged histone H2B (H2B-GFP) showed that hCdc14B was associated with chromatin during interphase and was released into the soluble fraction at prometaphase (Fig. 1A). Re-association with chromatin started around telophase and was completed after decondensation of chromatin in early G1 (Fig. 1A, right panels and bottom images). In contrast, virtually all hCdc14A was contained in the soluble cytoplasmic fraction throughout the cell cycle and was not detectable in the nucleoplasmic or chromatin fraction (Fig. 1A). The cell cycle-dependent, dynamic interaction of hCdc14B with nucleolar chromatin suggests that nucleolar sequestration serves a regulatory function in cycling cells similar to yeast Cdc14, restricting hCdc14B activity from early mitosis to telophase.


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)

Overexpression of hCdc14B alters progression through mitosis.A. hCdc14B is released from chromatin in early mitosis and is rebound in late M/early G1. HeLa cells expressing histone H2B-GFP (HeLa Kyoto) were arrested at G1/S by double thymidine-block (left) or at prometaphase of mitosis by nocodazole (right), and released for the indicated times. Release from nocodazole (nocod.) arrest was monitored visualizing H2B-GFP labelled chromatin (bottom panel). Synchronized cells were fractionated into cytoplasm (cyt.), nucleoplasm (nucl.) and chromatin (chr.), and analyzed on immunoblots for the proteins indicated (top panels). B. Overexpression of hCdc14B prolongs G2/M-phase. FACS of U2OS, U2OS-fl-hCdc14B and U2OS-fl-hCdc14BSL cells following release from G1/S (0 h) upon induction of hCdc14B expression by doxycyclin (Fig. S1). Cell numbers were calculated using the Cell Quest software program. The percentage of cells at the indicated cell cycle phases is presented. C. Overexpression of hCdc14B affects mitotic progression. Immunoblots showing the abundance of specific cell cycle marker proteins at distinct time points (h) after release from G1/S.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040744&req=5

pone-0014711-g001: Overexpression of hCdc14B alters progression through mitosis.A. hCdc14B is released from chromatin in early mitosis and is rebound in late M/early G1. HeLa cells expressing histone H2B-GFP (HeLa Kyoto) were arrested at G1/S by double thymidine-block (left) or at prometaphase of mitosis by nocodazole (right), and released for the indicated times. Release from nocodazole (nocod.) arrest was monitored visualizing H2B-GFP labelled chromatin (bottom panel). Synchronized cells were fractionated into cytoplasm (cyt.), nucleoplasm (nucl.) and chromatin (chr.), and analyzed on immunoblots for the proteins indicated (top panels). B. Overexpression of hCdc14B prolongs G2/M-phase. FACS of U2OS, U2OS-fl-hCdc14B and U2OS-fl-hCdc14BSL cells following release from G1/S (0 h) upon induction of hCdc14B expression by doxycyclin (Fig. S1). Cell numbers were calculated using the Cell Quest software program. The percentage of cells at the indicated cell cycle phases is presented. C. Overexpression of hCdc14B affects mitotic progression. Immunoblots showing the abundance of specific cell cycle marker proteins at distinct time points (h) after release from G1/S.
Mentions: Immunofluorescence studies have demonstrated that hCdc14B localizes within nucleoli in interphase cells [23], [28]. Fractionation of extracts from synchronized HeLa Kyoto cells that stably express GFP-tagged histone H2B (H2B-GFP) showed that hCdc14B was associated with chromatin during interphase and was released into the soluble fraction at prometaphase (Fig. 1A). Re-association with chromatin started around telophase and was completed after decondensation of chromatin in early G1 (Fig. 1A, right panels and bottom images). In contrast, virtually all hCdc14A was contained in the soluble cytoplasmic fraction throughout the cell cycle and was not detectable in the nucleoplasmic or chromatin fraction (Fig. 1A). The cell cycle-dependent, dynamic interaction of hCdc14B with nucleolar chromatin suggests that nucleolar sequestration serves a regulatory function in cycling cells similar to yeast Cdc14, restricting hCdc14B activity from early mitosis to telophase.

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