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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Published on: May 3, 2018

PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation

Seth S Margolis1, Susan Walsh, Douglas C Weiser

  • 1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

The EMBO Journal
|November 1, 2003
PubMed

Insights

Protein phosphatase 1 (PP1) dephosphorylates Cdc25 at a site suppressing its activity, enabling entry into M phase. This process involves Cdk2-dependent 14-3-3 removal and direct PP1-Cdc25 interaction.

Area of Science:

  • Cell cycle regulation
  • Molecular biology
  • Biochemistry

Background:

  • Protein phosphatase 1 (PP1) inhibition blocks M phase entry.
  • The specific PP1 substrate controlling this process remained unidentified.
  • Cdc25 phosphatase activity is crucial for mitotic entry.

Purpose of the Study:

  • Identify the PP1 substrate involved in M phase entry.
  • Elucidate the mechanism of Cdc25 regulation by PP1 and Cdk2.
  • Clarify the role of 14-3-3 proteins in Cdc25 regulation.

Main Methods:

  • Biochemical assays to study protein-protein interactions.
  • Phosphorylation and dephosphorylation site analysis.
  • In vitro kinase and phosphatase assays.

Main Results:

  • PP1 dephosphorylates Cdc25 at Ser287 (Xenopus) / Ser216 (human), a site suppressing interphase activity.
  • PP1 directly binds Cdc25 via an N-terminal motif.
  • 14-3-3 binding to phospho-Ser287 inhibits premature dephosphorylation.
  • Cdk2 activity is required for 14-3-3 removal from Cdc25, preceding dephosphorylation.
  • This reveals a Cdk2-dependent pathway for 14-3-3 dissociation.

Conclusions:

  • PP1 is essential for Cdc25 activation by dephosphorylating Ser287/216.
  • Cdk2-mediated 14-3-3 removal is a prerequisite for PP1-dependent Cdc25 activation.
  • These findings clarify Cdc25 activation steps and PP1's role in mitotic entry.

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