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Dual phosphorylation controls Cdc25 phosphatases and mitotic entry

Dmitry V Bulavin1, Yuichiro Higashimoto, Zoya N Demidenko

  • 1Gene Response Section, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Nature Cell Biology
|May 27, 2003
PubMed

Insights

Cdc25C protein phosphatase is regulated by phosphorylation. Serine 214 phosphorylation prevents Serine 216 binding, controlling cell cycle progression and mitotic entry.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cdc25C protein phosphatase activity is negatively regulated by phosphorylation at Serine 216, a key 14-3-3 binding site.
  • This regulation is crucial for blocking cell cycle progression into mitosis under normal conditions and after DNA damage.
  • During mitosis, Cdc25C is typically not phosphorylated at Serine 216, and ionizing radiation does not induce this phosphorylation or 14-3-3 binding.

Purpose of the Study:

  • To investigate the regulatory mechanisms of Cdc25C during mitosis.
  • To identify novel phosphorylation sites on Cdc25C that influence its activity and binding partners.
  • To elucidate the role of Serine 214 phosphorylation in controlling Cdc25C function and cell cycle progression.

Main Methods:

  • Site-directed mutagenesis to create Cdc25C mutants (S214A, S214A/S216A).
  • Introduction of exogenous Cdc25C mutants into HeLa cells depleted of endogenous Cdc25C.
  • Analysis of phosphorylation status, 14-3-3 binding, and mitotic entry timing.

Main Results:

  • Cdc25C is phosphorylated on Serine 214 during mitosis, which inhibits Serine 216 phosphorylation.
  • Mutation of Serine 214 to Alanine restores Serine 216 phosphorylation and 14-3-3 binding during mitosis.
  • Expression of Cdc25C(S214A) mutant delays mitotic entry, an effect dependent on Serine 216 phosphorylation.

Conclusions:

  • Mitotic phosphorylation of Cdc25C at Serine 214 acts as a negative regulator, preventing Serine 216 phosphorylation and 14-3-3 binding.
  • This pathway ensures Cdc25C remains active after mitosis initiation, maintaining proper cell cycle transitions.
  • A similar mechanism may regulate other mitotic phosphatases, including Cdc25B and orthologs in other species.

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