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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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Reduced activities of both cyclin A–Cdk2 and cyclin B1–Cdk1 in lysates of cells transfected with siRNA to Cdc25A or -B. (A) Delayed dephosphorylation of Cdk1 in cells treated with siRNA to Cdc25A or -B. SiRNA-transfected synchronized cells were subjected to Cdk1 immunoblotting. Arrows indicate the faster migrating unphosphorylated Cdk1 (bottom band) and the slower migrating phosphorylated Cdk1 (top band). siRNAs are indicated to the left. A quantification of the ratios of inactive versus active Cdk1 is available in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). (B) Reduced activation of cyclin A–Cdk2 in cells treated with siRNA to Cdc25A or -B. Cyclin A was immunoprecipitated from siRNA-transfected cells 9 h after release from thymidine block. The ability of the immunoprecipitate to phosphorylate histone H1 as well as the amount of Cdk2 in the immunoprecipitate was assessed. Bars show average from three independent experiments of normalized ratio between cyclin A–Cdk2 activity and amount of Cdk2.
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fig6: Reduced activities of both cyclin A–Cdk2 and cyclin B1–Cdk1 in lysates of cells transfected with siRNA to Cdc25A or -B. (A) Delayed dephosphorylation of Cdk1 in cells treated with siRNA to Cdc25A or -B. SiRNA-transfected synchronized cells were subjected to Cdk1 immunoblotting. Arrows indicate the faster migrating unphosphorylated Cdk1 (bottom band) and the slower migrating phosphorylated Cdk1 (top band). siRNAs are indicated to the left. A quantification of the ratios of inactive versus active Cdk1 is available in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). (B) Reduced activation of cyclin A–Cdk2 in cells treated with siRNA to Cdc25A or -B. Cyclin A was immunoprecipitated from siRNA-transfected cells 9 h after release from thymidine block. The ability of the immunoprecipitate to phosphorylate histone H1 as well as the amount of Cdk2 in the immunoprecipitate was assessed. Bars show average from three independent experiments of normalized ratio between cyclin A–Cdk2 activity and amount of Cdk2.

Mentions: Next, we sought to investigate the molecular mechanism behind the G2 delay caused by siRNA to Cdc25A and -B in our experiments. Cyclin B1–Cdk1 and cyclin A–Cdk2 are the main cyclin–Cdk complexes active at the G2/M transition (Pines and Hunter, 1989, 1990), and both can be dephosphorylated by Cdc25A and -B in vitro, whereas Cdc25C can only efficiently dephosphorylate cyclin B1–Cdk1 (Rudolph et al., 2001; Mailand et al., 2002). We analyzed the phosphorylation shift of Cdk1 by Western blot of synchronized siRNA-transfected cells. A slower migrating form of Cdk1, corresponding to the inactive kinase, appeared at the same time as cyclin B1, and followed the increase in cyclin B1 levels in intensity. Approximately 11 h after release from a thymidine block, the slower migrating form of Cdk1 disappeared in untransfected cells and in cells transfected with siRNA to Cdc25C, demonstrating Cdk1 activation. In contrast, inactive Cdk1 persisted in Cdc25A or -B siRNA-transfected cells (Fig. 6 A), suggesting that both Cdc25A and -B take part in the activation of Cdk1 at the G2/M transition. This is in agreement with previous studies (Gabrielli et al., 1997a; Lammer et al., 1998; Donzelli et al., 2002; Mailand et al., 2002).


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)

Reduced activities of both cyclin A–Cdk2 and cyclin B1–Cdk1 in lysates of cells transfected with siRNA to Cdc25A or -B. (A) Delayed dephosphorylation of Cdk1 in cells treated with siRNA to Cdc25A or -B. SiRNA-transfected synchronized cells were subjected to Cdk1 immunoblotting. Arrows indicate the faster migrating unphosphorylated Cdk1 (bottom band) and the slower migrating phosphorylated Cdk1 (top band). siRNAs are indicated to the left. A quantification of the ratios of inactive versus active Cdk1 is available in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). (B) Reduced activation of cyclin A–Cdk2 in cells treated with siRNA to Cdc25A or -B. Cyclin A was immunoprecipitated from siRNA-transfected cells 9 h after release from thymidine block. The ability of the immunoprecipitate to phosphorylate histone H1 as well as the amount of Cdk2 in the immunoprecipitate was assessed. Bars show average from three independent experiments of normalized ratio between cyclin A–Cdk2 activity and amount of Cdk2.
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Related In: Results  -  Collection

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fig6: Reduced activities of both cyclin A–Cdk2 and cyclin B1–Cdk1 in lysates of cells transfected with siRNA to Cdc25A or -B. (A) Delayed dephosphorylation of Cdk1 in cells treated with siRNA to Cdc25A or -B. SiRNA-transfected synchronized cells were subjected to Cdk1 immunoblotting. Arrows indicate the faster migrating unphosphorylated Cdk1 (bottom band) and the slower migrating phosphorylated Cdk1 (top band). siRNAs are indicated to the left. A quantification of the ratios of inactive versus active Cdk1 is available in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200503066/DC1). (B) Reduced activation of cyclin A–Cdk2 in cells treated with siRNA to Cdc25A or -B. Cyclin A was immunoprecipitated from siRNA-transfected cells 9 h after release from thymidine block. The ability of the immunoprecipitate to phosphorylate histone H1 as well as the amount of Cdk2 in the immunoprecipitate was assessed. Bars show average from three independent experiments of normalized ratio between cyclin A–Cdk2 activity and amount of Cdk2.
Mentions: Next, we sought to investigate the molecular mechanism behind the G2 delay caused by siRNA to Cdc25A and -B in our experiments. Cyclin B1–Cdk1 and cyclin A–Cdk2 are the main cyclin–Cdk complexes active at the G2/M transition (Pines and Hunter, 1989, 1990), and both can be dephosphorylated by Cdc25A and -B in vitro, whereas Cdc25C can only efficiently dephosphorylate cyclin B1–Cdk1 (Rudolph et al., 2001; Mailand et al., 2002). We analyzed the phosphorylation shift of Cdk1 by Western blot of synchronized siRNA-transfected cells. A slower migrating form of Cdk1, corresponding to the inactive kinase, appeared at the same time as cyclin B1, and followed the increase in cyclin B1 levels in intensity. Approximately 11 h after release from a thymidine block, the slower migrating form of Cdk1 disappeared in untransfected cells and in cells transfected with siRNA to Cdc25C, demonstrating Cdk1 activation. In contrast, inactive Cdk1 persisted in Cdc25A or -B siRNA-transfected cells (Fig. 6 A), suggesting that both Cdc25A and -B take part in the activation of Cdk1 at the G2/M transition. This is in agreement with previous studies (Gabrielli et al., 1997a; Lammer et al., 1998; Donzelli et al., 2002; Mailand et al., 2002).

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