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Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes
09:39

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Published on: December 20, 2014

Polo-like kinase 2-dependent phosphorylation of NPM/B23 on serine 4 triggers centriole duplication

Annekatrin Krause1, Ingrid Hoffmann

  • 1Cell Cycle Control and Carcinogenesis, German Cancer Research Center, Heidelberg, Germany.

Plos One
|March 31, 2010
PubMed

Insights

Polo-like kinase 2 (Plk2) interacts with Nucleophosmin (NPM/B23), directly regulating centrosome duplication. Plk2 phosphorylation of NPM/B23 on serine 4 is crucial for centriole reduplication during cell division.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Cancer Research

Background:

  • Centrosome duplication is tightly regulated for accurate cell division.
  • Deregulation leads to supernumerary centrosomes, aneuploidy, and is a hallmark of cancer.
  • Polo-like kinase 2 (Plk2) is known to regulate centrosome reproduction.

Purpose of the Study:

  • To identify novel Plk2 interacting proteins.
  • To investigate the role of Plk2 and its interaction with NPM/B23 in centrosome duplication.
  • To determine the functional significance of NPM/B23 phosphorylation by Plk2.

Main Methods:

  • In vitro and in vivo protein-protein interaction assays.
  • Identification of Plk2 binding partners.
  • Site-directed mutagenesis to create NPM/B23 phosphorylation mutants (S4A and phospho-mimicking).
  • Analysis of centriole duplication in cell cycle-arrested and unperturbed cells expressing NPM/B23 mutants.

Main Results:

  • Nucleophosmin (NPM/B23) was identified as a novel binding partner of Plk2.
  • Plk2 and NPM/B23 interact both in vitro and in vivo.
  • Plk2 directly phosphorylates NPM/B23 on serine 4 during S-phase.
  • Mutational analysis revealed that NPM/B23 phosphorylation is essential for centriole reduplication.

Conclusions:

  • NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication.
  • Phosphorylation of NPM/B23 on serine 4 by Plk2 is a key event that triggers or regulates centriole duplication.
  • This finding provides new insights into the molecular mechanisms controlling centrosome duplication and its potential role in cancer.

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