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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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The PHLPP1 N-Terminal Extension Is a Mitotic Cdk1 Substrate and Controls an Interactome Switch

Agnieszka T Kawashima1,2, Cassandra Wong3, Gema Lordén1

  • 1Department of Pharmacology, University of California, San Diego, California, USA.

Molecular and Cellular Biology
|January 5, 2021
PubMed

Insights

PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is regulated during mitosis. Its phosphorylation controls binding to mitotic proteins, ensuring accurate cell division and supporting its tumor suppressor role.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Cancer Research

Background:

  • PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) acts as a tumor suppressor by dephosphorylating Akt.
  • The molecular mechanisms regulating PHLPP1 activity, particularly during the cell cycle, remain largely uncharacterized.

Purpose of the Study:

  • To investigate the cell cycle-dependent regulation of PHLPP1.
  • To elucidate the role of PHLPP1 phosphorylation and its interaction partners during mitosis.

Main Methods:

  • Cell cycle analysis to assess mitotic delays and chromosomal segregation errors.
  • Proximity-dependent biotin identification (BioID) to identify PHLPP1 interacting proteins during mitosis.
  • Site-directed mutagenesis to study the role of the N-terminal extension (NTE).

Main Results:

  • PHLPP1 undergoes cell cycle-dependent regulation, with deletion causing mitotic delays and increased chromosomal segregation errors.
  • PHLPP1 is hyperphosphorylated during mitosis by Cdk1 within its N-terminal extension (NTE).
  • Mitotic PHLPP1 dissociates from plasma membrane scaffolds (e.g., Scribble) and interacts with kinetochore/mitotic spindle proteins (e.g., KNL1, TPX2) in an NTE-dependent manner.

Conclusions:

  • Mitotic phosphorylation of PHLPP1 by Cdk1 regulates its binding to mitotic partners, facilitating accurate cell cycle progression.
  • These findings reveal a novel mechanism for PHLPP1 regulation and suggest its role in maintaining genomic stability, relevant to its tumor-suppressive function.
Keywords:
Cdk1PHLPPmitosisphosphatase

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