Abstract

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.

Highlights

  • Human cells blocks mitotic entry due to impaired assembly of Cdk1-cyclin B complexes, indicating that the activating phosphorylation is a prerequisite for formation of stable complexes in vivo [8]

  • Overexpression of Cdc25A Leads to an Accelerated G2/M Transition—Recent publications point to a potential importance of Cdc25A in mitosis (20 –22)

  • In order to further investigate the role of Cdc25A phosphatase in an unperturbed cell cycle, we made use of a stable inducible U2OS cell line, expressing Cdc25A in a tetracycline-dependent manner

Read more

Summary

Introduction

Human cells blocks mitotic entry due to impaired assembly of Cdk1-cyclin B complexes, indicating that the activating phosphorylation is a prerequisite for formation of stable complexes in vivo [8]. This accumulation of Tyr15-phosphorylated Cdk1 in Cdc25A-overexpressing cells was surprising because it is anticipated that Cdc25 phosphatases activate Cdk1 via removal of inhibitory phosphates from both Thr14 and Tyr15 residues.

Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call