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Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome.

Lindqvist A, Källström H, Lundgren A, Barsoum E, Rosenthal CK - J. Cell Biol. (2005)

Bottom Line: Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear.Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis.We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden.

ABSTRACT
Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.

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Related in: MedlinePlus

Combining Cdc25A and -B siRNA leads to a cell cycle block. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam). Samples were harvested for FACS analysis at the indicated time points after release from a thymidine block. The left graph shows the combined fractions of cells in G1 and S phases, and the right graph shows cells in G2 or M phases.
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fig2: Combining Cdc25A and -B siRNA leads to a cell cycle block. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam). Samples were harvested for FACS analysis at the indicated time points after release from a thymidine block. The left graph shows the combined fractions of cells in G1 and S phases, and the right graph shows cells in G2 or M phases.

Mentions: SiRNA targeting of Cdc25A or -B caused a delay, but not a block of the cell cycle, which may indicate some redundancy between the isoforms. Therefore, we were interested in knowing the effect of suppressing the expression of more than one Cdc25 in the cell. In an experiment similar to the ones depicted in Fig. 1, we targeted the three Cdc25 homologues in all possible combinations. Combining siRNA to Cdc25C with siRNA to either Cdc25A or -B did not further increase the time of the delay seen when targeting Cdc25A or -B separately (unpublished data). Simultaneous targeting of Cdc25A and -B, however, caused a more prominent G2/M accumulation than targeting Cdc25A and -B individually (unsynchronized cells, Fig. 1 B; synchronized cells, Fig. 2). In fact, very few of the cells cotransfected with siRNA to Cdc25A and -B were able to go through mitosis and enter the next cell cycle during the time course of the experiment (Fig. 2). This suggests that Cdc25A and -B cooperate, and at least partly can compensate for each other, in catalyzing entry into mitosis.


Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome.

Lindqvist A, Källström H, Lundgren A, Barsoum E, Rosenthal CK - J. Cell Biol. (2005)

Combining Cdc25A and -B siRNA leads to a cell cycle block. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam). Samples were harvested for FACS analysis at the indicated time points after release from a thymidine block. The left graph shows the combined fractions of cells in G1 and S phases, and the right graph shows cells in G2 or M phases.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171226&req=5

fig2: Combining Cdc25A and -B siRNA leads to a cell cycle block. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam). Samples were harvested for FACS analysis at the indicated time points after release from a thymidine block. The left graph shows the combined fractions of cells in G1 and S phases, and the right graph shows cells in G2 or M phases.
Mentions: SiRNA targeting of Cdc25A or -B caused a delay, but not a block of the cell cycle, which may indicate some redundancy between the isoforms. Therefore, we were interested in knowing the effect of suppressing the expression of more than one Cdc25 in the cell. In an experiment similar to the ones depicted in Fig. 1, we targeted the three Cdc25 homologues in all possible combinations. Combining siRNA to Cdc25C with siRNA to either Cdc25A or -B did not further increase the time of the delay seen when targeting Cdc25A or -B separately (unpublished data). Simultaneous targeting of Cdc25A and -B, however, caused a more prominent G2/M accumulation than targeting Cdc25A and -B individually (unsynchronized cells, Fig. 1 B; synchronized cells, Fig. 2). In fact, very few of the cells cotransfected with siRNA to Cdc25A and -B were able to go through mitosis and enter the next cell cycle during the time course of the experiment (Fig. 2). This suggests that Cdc25A and -B cooperate, and at least partly can compensate for each other, in catalyzing entry into mitosis.

Bottom Line: Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear.Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis.We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden.

ABSTRACT
Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.

Show MeSH
Related in: MedlinePlus