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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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Cyclin accumulation is delayed in cells injected with siRNA to Cdc25A or -B. HeLa cells were microinjected with pCFP-Golgi as a marker for injected cells, together with siRNA to Cdc25A, -B, or -C, and fixed 8 h after release from a thymidine block. Cells were stained with antibodies to cyclin A, cyclin B1, and Cdc25B and the fluorescence was quantified as described in Materials and methods. (A) Example of images (maximum intensity projections) of cells injected with siRNA to Cdc25B. Microinjected cells (which express CFP-Golgi) are indicated by arrows. Bar, 10 μm. (B) Average fluorescence levels of Cdc25B (left), cyclin A (middle), and cyclin B1 (right) in siRNA-treated cells. The horizontal lines show the average, whereas the vertical lines visualize the quartiles of the quantified fluorescence. (C) Cyclin A and B1 fluorescence in single cells. Nuclear cyclin A levels (x axis) plotted against cytoplasmic cyclin B1 levels (y axis) for individual cells. In each graph the injected siRNA and number of quantified cells are shown. To facilitate the comparison, the area that includes all uninjected cells is marked in red and transferred to all graphs. As shown, a subset of cells injected with siRNA to Cdc25B express very little cyclin B1 but contain high cyclin A levels. NI, not injected.
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fig4: Cyclin accumulation is delayed in cells injected with siRNA to Cdc25A or -B. HeLa cells were microinjected with pCFP-Golgi as a marker for injected cells, together with siRNA to Cdc25A, -B, or -C, and fixed 8 h after release from a thymidine block. Cells were stained with antibodies to cyclin A, cyclin B1, and Cdc25B and the fluorescence was quantified as described in Materials and methods. (A) Example of images (maximum intensity projections) of cells injected with siRNA to Cdc25B. Microinjected cells (which express CFP-Golgi) are indicated by arrows. Bar, 10 μm. (B) Average fluorescence levels of Cdc25B (left), cyclin A (middle), and cyclin B1 (right) in siRNA-treated cells. The horizontal lines show the average, whereas the vertical lines visualize the quartiles of the quantified fluorescence. (C) Cyclin A and B1 fluorescence in single cells. Nuclear cyclin A levels (x axis) plotted against cytoplasmic cyclin B1 levels (y axis) for individual cells. In each graph the injected siRNA and number of quantified cells are shown. To facilitate the comparison, the area that includes all uninjected cells is marked in red and transferred to all graphs. As shown, a subset of cells injected with siRNA to Cdc25B express very little cyclin B1 but contain high cyclin A levels. NI, not injected.

Mentions: It was previously reported that suppression of Cdc25A protein expression leads to a reduction of cyclin A levels (Turowski et al., 2003), which caused us to more carefully monitor by immunofluorescence the accumulation of cyclin A and B1 in siRNA-treated cells. We microinjected cells with siRNA together with a plasmid to mark microinjected cells. The cells were then synchronized, fixed, and stained with antibodies to cyclin A and B1, as well as with Cdc25B antibodies, to monitor the efficiency of the RNAi treatment. The protocol for acquiring comparable images for quantification is described in Materials and methods. The fluorescence of between 46 and 77 cells was measured for each siRNA that was microinjected. Fig. 4 B shows the average fluorescence of Cdc25B, cyclin A, and cyclin B1 in siRNA-treated cells. We noticed that cyclin B1 levels were reduced in cells treated with either Cdc25A or -B siRNA, but not in cells treated with siRNA to Cdc25C or CD46 (control) or in untreated cells (Fig. 4 B). Also, average cyclin A levels were slightly lower in cells treated with Cdc25A siRNA. As expected, Cdc25B levels were greatly reduced in cells microinjected with siRNA to Cdc25B.


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)

Cyclin accumulation is delayed in cells injected with siRNA to Cdc25A or -B. HeLa cells were microinjected with pCFP-Golgi as a marker for injected cells, together with siRNA to Cdc25A, -B, or -C, and fixed 8 h after release from a thymidine block. Cells were stained with antibodies to cyclin A, cyclin B1, and Cdc25B and the fluorescence was quantified as described in Materials and methods. (A) Example of images (maximum intensity projections) of cells injected with siRNA to Cdc25B. Microinjected cells (which express CFP-Golgi) are indicated by arrows. Bar, 10 μm. (B) Average fluorescence levels of Cdc25B (left), cyclin A (middle), and cyclin B1 (right) in siRNA-treated cells. The horizontal lines show the average, whereas the vertical lines visualize the quartiles of the quantified fluorescence. (C) Cyclin A and B1 fluorescence in single cells. Nuclear cyclin A levels (x axis) plotted against cytoplasmic cyclin B1 levels (y axis) for individual cells. In each graph the injected siRNA and number of quantified cells are shown. To facilitate the comparison, the area that includes all uninjected cells is marked in red and transferred to all graphs. As shown, a subset of cells injected with siRNA to Cdc25B express very little cyclin B1 but contain high cyclin A levels. NI, not injected.
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fig4: Cyclin accumulation is delayed in cells injected with siRNA to Cdc25A or -B. HeLa cells were microinjected with pCFP-Golgi as a marker for injected cells, together with siRNA to Cdc25A, -B, or -C, and fixed 8 h after release from a thymidine block. Cells were stained with antibodies to cyclin A, cyclin B1, and Cdc25B and the fluorescence was quantified as described in Materials and methods. (A) Example of images (maximum intensity projections) of cells injected with siRNA to Cdc25B. Microinjected cells (which express CFP-Golgi) are indicated by arrows. Bar, 10 μm. (B) Average fluorescence levels of Cdc25B (left), cyclin A (middle), and cyclin B1 (right) in siRNA-treated cells. The horizontal lines show the average, whereas the vertical lines visualize the quartiles of the quantified fluorescence. (C) Cyclin A and B1 fluorescence in single cells. Nuclear cyclin A levels (x axis) plotted against cytoplasmic cyclin B1 levels (y axis) for individual cells. In each graph the injected siRNA and number of quantified cells are shown. To facilitate the comparison, the area that includes all uninjected cells is marked in red and transferred to all graphs. As shown, a subset of cells injected with siRNA to Cdc25B express very little cyclin B1 but contain high cyclin A levels. NI, not injected.
Mentions: It was previously reported that suppression of Cdc25A protein expression leads to a reduction of cyclin A levels (Turowski et al., 2003), which caused us to more carefully monitor by immunofluorescence the accumulation of cyclin A and B1 in siRNA-treated cells. We microinjected cells with siRNA together with a plasmid to mark microinjected cells. The cells were then synchronized, fixed, and stained with antibodies to cyclin A and B1, as well as with Cdc25B antibodies, to monitor the efficiency of the RNAi treatment. The protocol for acquiring comparable images for quantification is described in Materials and methods. The fluorescence of between 46 and 77 cells was measured for each siRNA that was microinjected. Fig. 4 B shows the average fluorescence of Cdc25B, cyclin A, and cyclin B1 in siRNA-treated cells. We noticed that cyclin B1 levels were reduced in cells treated with either Cdc25A or -B siRNA, but not in cells treated with siRNA to Cdc25C or CD46 (control) or in untreated cells (Fig. 4 B). Also, average cyclin A levels were slightly lower in cells treated with Cdc25A siRNA. As expected, Cdc25B levels were greatly reduced in cells microinjected with siRNA to Cdc25B.

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