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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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Cells with reduced Cdc25A or -B levels are delayed in G2/M progression. (A) Specific siRNA targeting of Cdc25 isoforms. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam) in the first release of a double thymidine block. 9 h after the second release (40 h after transfection), cells were harvested for Western blot. The antibodies used for immunoblotting are indicated to the left of and transfected siRNAs below the figure. The levels of Lamin B (arrow) indicate equal protein loading (not affected by Lamin A/C silencing). NT, not transfected. (B) FACS profiles of unsynchronized cells 24, 48, and 72 h after transfection of siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam). The x axis is logarithmic. (C) Outline of experimental setup used when transfecting siRNA for collection of synchronized cells at different time points. Cells were subjected to either a single or a double thymidine block. (D) FACS profiles of synchronized cells. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), or Lamin A/C (Lam). Samples were taken for FACS analysis at 0, 6, and 12 h after release from a thymidine block. The x axis is linear. (E) Time-lapse microscopy of synchronized cells. SiRNA-injected HeLa or HEK293 cells were followed with time-lapse microscopy after release from a thymidine block. The timing of mitosis of microinjected cells was compared with the timing of mitosis of uninjected cells in the same dish. The siRNA used for injection and the number of monitored cells is indicated in the graphs. Two different sets of siRNA oligos were used (A, B, and C; A2, B2, and C2). For an example of images from time-lapse microscopy, see Fig. 3 B.
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fig1: Cells with reduced Cdc25A or -B levels are delayed in G2/M progression. (A) Specific siRNA targeting of Cdc25 isoforms. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam) in the first release of a double thymidine block. 9 h after the second release (40 h after transfection), cells were harvested for Western blot. The antibodies used for immunoblotting are indicated to the left of and transfected siRNAs below the figure. The levels of Lamin B (arrow) indicate equal protein loading (not affected by Lamin A/C silencing). NT, not transfected. (B) FACS profiles of unsynchronized cells 24, 48, and 72 h after transfection of siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam). The x axis is logarithmic. (C) Outline of experimental setup used when transfecting siRNA for collection of synchronized cells at different time points. Cells were subjected to either a single or a double thymidine block. (D) FACS profiles of synchronized cells. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), or Lamin A/C (Lam). Samples were taken for FACS analysis at 0, 6, and 12 h after release from a thymidine block. The x axis is linear. (E) Time-lapse microscopy of synchronized cells. SiRNA-injected HeLa or HEK293 cells were followed with time-lapse microscopy after release from a thymidine block. The timing of mitosis of microinjected cells was compared with the timing of mitosis of uninjected cells in the same dish. The siRNA used for injection and the number of monitored cells is indicated in the graphs. Two different sets of siRNA oligos were used (A, B, and C; A2, B2, and C2). For an example of images from time-lapse microscopy, see Fig. 3 B.

Mentions: All three human Cdc25 phosphatases are suggested to play a role in mitosis. To evaluate the role of the individual Cdc25 isoforms in mitotic entry, we used RNAi to reduce the levels of Cdc25A, -B, and -C in HeLa and HEK293 cells. The siRNA oligo sequences were designed to target all splice versions of each Cdc25 homologue. Western blots on whole cell lysates of G2 HeLa cells, 40 h after transfection with siRNA, showed that the protein level of each Cdc25 could be specifically and efficiently reduced by the siRNA treatment (Fig. 1 A).


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)

Cells with reduced Cdc25A or -B levels are delayed in G2/M progression. (A) Specific siRNA targeting of Cdc25 isoforms. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam) in the first release of a double thymidine block. 9 h after the second release (40 h after transfection), cells were harvested for Western blot. The antibodies used for immunoblotting are indicated to the left of and transfected siRNAs below the figure. The levels of Lamin B (arrow) indicate equal protein loading (not affected by Lamin A/C silencing). NT, not transfected. (B) FACS profiles of unsynchronized cells 24, 48, and 72 h after transfection of siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam). The x axis is logarithmic. (C) Outline of experimental setup used when transfecting siRNA for collection of synchronized cells at different time points. Cells were subjected to either a single or a double thymidine block. (D) FACS profiles of synchronized cells. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), or Lamin A/C (Lam). Samples were taken for FACS analysis at 0, 6, and 12 h after release from a thymidine block. The x axis is linear. (E) Time-lapse microscopy of synchronized cells. SiRNA-injected HeLa or HEK293 cells were followed with time-lapse microscopy after release from a thymidine block. The timing of mitosis of microinjected cells was compared with the timing of mitosis of uninjected cells in the same dish. The siRNA used for injection and the number of monitored cells is indicated in the graphs. Two different sets of siRNA oligos were used (A, B, and C; A2, B2, and C2). For an example of images from time-lapse microscopy, see Fig. 3 B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171226&req=5

fig1: Cells with reduced Cdc25A or -B levels are delayed in G2/M progression. (A) Specific siRNA targeting of Cdc25 isoforms. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam) in the first release of a double thymidine block. 9 h after the second release (40 h after transfection), cells were harvested for Western blot. The antibodies used for immunoblotting are indicated to the left of and transfected siRNAs below the figure. The levels of Lamin B (arrow) indicate equal protein loading (not affected by Lamin A/C silencing). NT, not transfected. (B) FACS profiles of unsynchronized cells 24, 48, and 72 h after transfection of siRNA to Cdc25A (A), -B (B), -C (C), -A and -B (AB), or Lamin A/C (Lam). The x axis is logarithmic. (C) Outline of experimental setup used when transfecting siRNA for collection of synchronized cells at different time points. Cells were subjected to either a single or a double thymidine block. (D) FACS profiles of synchronized cells. HeLa cells were transfected with siRNA to Cdc25A (A), -B (B), -C (C), or Lamin A/C (Lam). Samples were taken for FACS analysis at 0, 6, and 12 h after release from a thymidine block. The x axis is linear. (E) Time-lapse microscopy of synchronized cells. SiRNA-injected HeLa or HEK293 cells were followed with time-lapse microscopy after release from a thymidine block. The timing of mitosis of microinjected cells was compared with the timing of mitosis of uninjected cells in the same dish. The siRNA used for injection and the number of monitored cells is indicated in the graphs. Two different sets of siRNA oligos were used (A, B, and C; A2, B2, and C2). For an example of images from time-lapse microscopy, see Fig. 3 B.
Mentions: All three human Cdc25 phosphatases are suggested to play a role in mitosis. To evaluate the role of the individual Cdc25 isoforms in mitotic entry, we used RNAi to reduce the levels of Cdc25A, -B, and -C in HeLa and HEK293 cells. The siRNA oligo sequences were designed to target all splice versions of each Cdc25 homologue. Western blots on whole cell lysates of G2 HeLa cells, 40 h after transfection with siRNA, showed that the protein level of each Cdc25 could be specifically and efficiently reduced by the siRNA treatment (Fig. 1 A).

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