Cdc25 phosphatases are required for timely assembly of CDK1-cyclin B at the G2/M transition.
Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr(161) by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr(14) and Tyr(15) phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G(2)/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr(15). In addition, Tyr(15)-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G(2) and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr(161) phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G(2)/M is tightly coupled and regulated by Cdc25 phosphatases.
Timofeev
Oleg
O
Cell Cycle Control and Carcinogenesis, German Cancer Research Center, F045, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany.
Cizmecioglu
Onur
O
Settele
Florian
F
Kempf
Tore
T
Hoffmann
Ingrid
I
D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D019926:Cyclin B / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D016195:G2 Phase; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014443:Tyrosine / Q000737:chemistry; D020687:cdc25 Phosphatases / Q000378:metabolism*; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D019926:Cyclin B; D014443:Tyrosine; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases; D020687:cdc25 Phosphatases; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human