Limits...
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.

Show MeSH

Related in: MedlinePlus

Cdc25B dephosphorylates cyclin B1–Cdk1 on the centrosomes. HeLa cells were microinjected with pCFP-Golgi together with siRNA to Cdc25A, -B, -C, or CD46 after release of a double thymidine block and fixed 8 or 12 h after release. Cells were stained with antibodies to phosphorylated Cdk1 (Y15P Cdk1) and to cyclin B1. The specificity of the Cdk1-P antibody is demonstrated in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). (A) Examples of cells microinjected with siRNA to Cdc25B and stained with Y15P Cdk1 12 h after release from thymidine block. The Y15P Cdk1 staining is high on centrosomes in G2 cells but low in metaphase cells. Most surrounding cells have passed through mitosis. Bar, 25 μm. (B) Cytoplasmic cyclin B1 (x axis) and Y15P Cdk1 (y axis) fluorescence 8 h after release from a thymidine block. Each dot corresponds to one cell. The diagonal line indicates the levels of cyclin B1 and Y15P at which the control cells are starting to activate Cdk1 (the Y15P Cdk1 accumulation slows down). The fraction of cells above this line is indicated in percent and in numbers of cells in each graph. Cytoplasmic (C) and centrosomal (D) cyclin B1 (x axis) and Y15P Cdk1 (y axis) fluorescence in G2 cells 12 h after release from a thymidine block. Red dots represent cells with separated centrosomes and blue dots cells where centrosomes are not separated. The diagonal line indicates the levels of cyclin B1 and Y15P above which the majority of control cells contain separated centrosomes. The fraction of cells with unseparated centrosomes above this line is indicated in percent and in numbers of cells in each graph. Note that cells situated above the diagonal line in B have not separated their centrosomes and most probably correspond to cells below the diagonal line in C.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171226&req=5

fig7: Cdc25B dephosphorylates cyclin B1–Cdk1 on the centrosomes. HeLa cells were microinjected with pCFP-Golgi together with siRNA to Cdc25A, -B, -C, or CD46 after release of a double thymidine block and fixed 8 or 12 h after release. Cells were stained with antibodies to phosphorylated Cdk1 (Y15P Cdk1) and to cyclin B1. The specificity of the Cdk1-P antibody is demonstrated in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). (A) Examples of cells microinjected with siRNA to Cdc25B and stained with Y15P Cdk1 12 h after release from thymidine block. The Y15P Cdk1 staining is high on centrosomes in G2 cells but low in metaphase cells. Most surrounding cells have passed through mitosis. Bar, 25 μm. (B) Cytoplasmic cyclin B1 (x axis) and Y15P Cdk1 (y axis) fluorescence 8 h after release from a thymidine block. Each dot corresponds to one cell. The diagonal line indicates the levels of cyclin B1 and Y15P at which the control cells are starting to activate Cdk1 (the Y15P Cdk1 accumulation slows down). The fraction of cells above this line is indicated in percent and in numbers of cells in each graph. Cytoplasmic (C) and centrosomal (D) cyclin B1 (x axis) and Y15P Cdk1 (y axis) fluorescence in G2 cells 12 h after release from a thymidine block. Red dots represent cells with separated centrosomes and blue dots cells where centrosomes are not separated. The diagonal line indicates the levels of cyclin B1 and Y15P above which the majority of control cells contain separated centrosomes. The fraction of cells with unseparated centrosomes above this line is indicated in percent and in numbers of cells in each graph. Note that cells situated above the diagonal line in B have not separated their centrosomes and most probably correspond to cells below the diagonal line in C.

Mentions: To evaluate the Cdk1-P antibody in immunofluorescence, we first stained cells not treated with any siRNA. The Cdk1-P staining appeared in the cytoplasm and on the centrosomes together with cyclin B1 staining in G2 cells but was close to background levels in metaphase cells, showing that the antibody does not recognize unphosphorylated Cdk1 in immunofluorescence (Fig. 7 A and Fig. S2 D, available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). We then plotted the quantified Cdk-P fluorescence against the cyclin B1 fluorescence signal. In uninjected cells or in cells treated with a control siRNA, the cytoplasmic Cdk1-P staining increased as cyclin B1 levels increased (Fig. 7 B, left). This indicates that as the cyclin B1–Cdk1 complex accumulates, Cdk1 is inactivated by phosphorylation as expected. Thus, we believe that the Cdk1-P antibody staining correctly represents the pool of inactive cyclin B1–Cdk1 in the cell. When cells microinjected with siRNA to Cdc25A or -B were observed 8 h after release from the thymidine block, the accumulation of Cdk1-P was observed to be similar to that in control cells both in the cytoplasm (Fig. 7 B) and on centrosomes (not depicted). However, the cyclin B1 accumulation was delayed in these cells, confirming our previous finding (Fig. 5 B). Thus, 8 h after release from a thymidine block, the siRNA-injected cells did not differ in Cdk1 Y15 phosphorylation but, rather, in the amount of cyclin B1–Cdk1 complexes. Therefore, we turned our focus to a later time point, when cyclin B1 had accumulated. 12 h after release from a thymidine block, most of the cells injected with siRNA to Cdc25C had entered mitosis, whereas a large portion of cells injected with siRNA to Cdc25A or -B had still not divided (Fig. 7 A and not depicted). We quantified cytoplasmic as well as centrosomal cyclin B1 and Cdk1-P immunofluorescence signals and measured the distance between the centrosomes in the same cell. Centrosomes were considered separated when the distance between them exceeded 2 μm.


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)

Cdc25B dephosphorylates cyclin B1–Cdk1 on the centrosomes. HeLa cells were microinjected with pCFP-Golgi together with siRNA to Cdc25A, -B, -C, or CD46 after release of a double thymidine block and fixed 8 or 12 h after release. Cells were stained with antibodies to phosphorylated Cdk1 (Y15P Cdk1) and to cyclin B1. The specificity of the Cdk1-P antibody is demonstrated in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). (A) Examples of cells microinjected with siRNA to Cdc25B and stained with Y15P Cdk1 12 h after release from thymidine block. The Y15P Cdk1 staining is high on centrosomes in G2 cells but low in metaphase cells. Most surrounding cells have passed through mitosis. Bar, 25 μm. (B) Cytoplasmic cyclin B1 (x axis) and Y15P Cdk1 (y axis) fluorescence 8 h after release from a thymidine block. Each dot corresponds to one cell. The diagonal line indicates the levels of cyclin B1 and Y15P at which the control cells are starting to activate Cdk1 (the Y15P Cdk1 accumulation slows down). The fraction of cells above this line is indicated in percent and in numbers of cells in each graph. Cytoplasmic (C) and centrosomal (D) cyclin B1 (x axis) and Y15P Cdk1 (y axis) fluorescence in G2 cells 12 h after release from a thymidine block. Red dots represent cells with separated centrosomes and blue dots cells where centrosomes are not separated. The diagonal line indicates the levels of cyclin B1 and Y15P above which the majority of control cells contain separated centrosomes. The fraction of cells with unseparated centrosomes above this line is indicated in percent and in numbers of cells in each graph. Note that cells situated above the diagonal line in B have not separated their centrosomes and most probably correspond to cells below the diagonal line in C.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Cdc25B dephosphorylates cyclin B1–Cdk1 on the centrosomes. HeLa cells were microinjected with pCFP-Golgi together with siRNA to Cdc25A, -B, -C, or CD46 after release of a double thymidine block and fixed 8 or 12 h after release. Cells were stained with antibodies to phosphorylated Cdk1 (Y15P Cdk1) and to cyclin B1. The specificity of the Cdk1-P antibody is demonstrated in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). (A) Examples of cells microinjected with siRNA to Cdc25B and stained with Y15P Cdk1 12 h after release from thymidine block. The Y15P Cdk1 staining is high on centrosomes in G2 cells but low in metaphase cells. Most surrounding cells have passed through mitosis. Bar, 25 μm. (B) Cytoplasmic cyclin B1 (x axis) and Y15P Cdk1 (y axis) fluorescence 8 h after release from a thymidine block. Each dot corresponds to one cell. The diagonal line indicates the levels of cyclin B1 and Y15P at which the control cells are starting to activate Cdk1 (the Y15P Cdk1 accumulation slows down). The fraction of cells above this line is indicated in percent and in numbers of cells in each graph. Cytoplasmic (C) and centrosomal (D) cyclin B1 (x axis) and Y15P Cdk1 (y axis) fluorescence in G2 cells 12 h after release from a thymidine block. Red dots represent cells with separated centrosomes and blue dots cells where centrosomes are not separated. The diagonal line indicates the levels of cyclin B1 and Y15P above which the majority of control cells contain separated centrosomes. The fraction of cells with unseparated centrosomes above this line is indicated in percent and in numbers of cells in each graph. Note that cells situated above the diagonal line in B have not separated their centrosomes and most probably correspond to cells below the diagonal line in C.
Mentions: To evaluate the Cdk1-P antibody in immunofluorescence, we first stained cells not treated with any siRNA. The Cdk1-P staining appeared in the cytoplasm and on the centrosomes together with cyclin B1 staining in G2 cells but was close to background levels in metaphase cells, showing that the antibody does not recognize unphosphorylated Cdk1 in immunofluorescence (Fig. 7 A and Fig. S2 D, available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). We then plotted the quantified Cdk-P fluorescence against the cyclin B1 fluorescence signal. In uninjected cells or in cells treated with a control siRNA, the cytoplasmic Cdk1-P staining increased as cyclin B1 levels increased (Fig. 7 B, left). This indicates that as the cyclin B1–Cdk1 complex accumulates, Cdk1 is inactivated by phosphorylation as expected. Thus, we believe that the Cdk1-P antibody staining correctly represents the pool of inactive cyclin B1–Cdk1 in the cell. When cells microinjected with siRNA to Cdc25A or -B were observed 8 h after release from the thymidine block, the accumulation of Cdk1-P was observed to be similar to that in control cells both in the cytoplasm (Fig. 7 B) and on centrosomes (not depicted). However, the cyclin B1 accumulation was delayed in these cells, confirming our previous finding (Fig. 5 B). Thus, 8 h after release from a thymidine block, the siRNA-injected cells did not differ in Cdk1 Y15 phosphorylation but, rather, in the amount of cyclin B1–Cdk1 complexes. Therefore, we turned our focus to a later time point, when cyclin B1 had accumulated. 12 h after release from a thymidine block, most of the cells injected with siRNA to Cdc25C had entered mitosis, whereas a large portion of cells injected with siRNA to Cdc25A or -B had still not divided (Fig. 7 A and not depicted). We quantified cytoplasmic as well as centrosomal cyclin B1 and Cdk1-P immunofluorescence signals and measured the distance between the centrosomes in the same cell. Centrosomes were considered separated when the distance between them exceeded 2 μm.

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