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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Multisite phosphorylation of nuclear interaction partner of ALK (NIPA) at G2/M involves cyclin B1/Cdk1

Florian Bassermann1, Christine von Klitzing, Anna Lena Illert

  • 1Department of Internal Medicine III, Technical University of Munich, 81675 Munich, Germany, and Department of Pathology, St. Jude Chidren's Research Hospital, Memphis, TN 38105, USA. florian.bassermann@med.nyu.edu

The Journal of Biological Chemistry
|March 29, 2007
PubMed

Insights

Nuclear interaction partner of ALK (NIPA) regulates cell division by targeting cyclin B1. Phosphorylation of NIPA by cyclin B1/Cdk1 in mitosis controls this process, ensuring proper cell cycle progression.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Nuclear interaction partner of ALK (NIPA) is an F-box protein forming the SCFNIPA ubiquitin E3 ligase complex.
  • SCFNIPA regulates mitotic entry by targeting nuclear cyclin B1 for ubiquitination during interphase.
  • NIPA phosphorylation in late G2/M phases inactivates SCFNIPA, allowing cyclin B1 accumulation.

Purpose of the Study:

  • Identify the NIPA region responsible for cyclin B1 binding.
  • Characterize novel phosphorylation sites on NIPA crucial for G2/M regulation.
  • Investigate the role of cyclin B1/Cdk1 in NIPA phosphorylation and SCFNIPA complex inactivation.

Main Methods:

  • Protein interaction mapping to identify NIPA-cyclin B1 binding region.
  • Site-directed mutagenesis to analyze NIPA phosphorylation site function.
  • Western blotting and immunoprecipitation to assess protein levels and complex activity.

Main Results:

  • Specific NIPA region mediating cyclin B1 binding identified.
  • Serine residues 359 and 395 identified as critical G2/M phosphorylation sites on NIPA.
  • Mutation of Ser-359 and Ser-395 impaired SCFNIPA inactivation, reducing mitotic cyclin B1 levels.
  • Cyclin B1/Cdk1 was found to phosphorylate NIPA at Ser-395 during mitosis.

Conclusions:

  • NIPA phosphorylation by cyclin B1/Cdk1 is a sequential process that regulates SCFNIPA activity.
  • Cyclin B1/Cdk1 amplifies NIPA phosphorylation, contributing to the regulation of its own abundance in early mitosis.
  • These findings provide insights into the intricate feedback mechanisms controlling cell cycle progression.

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