Related Experiment Video

Updated: May 14, 2025

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

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Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing

Henry Y Ng1, Devon H Whelpley1, Armin N Adly1

  • 1Department of Physiology, University of California San Francisco, San Francisco, CA, USA.

Nature Communications
|May 9, 2025
PubMed

Insights

A novel phosphate-binding pocket on B-type cyclins, similar to Cks1, regulates multisite phosphorylation of cyclin-dependent kinase (CDK) substrates. This finding is crucial for understanding cell cycle control and robust timing of cell division.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cell cycle progression relies on cyclin-dependent kinases (CDKs) and their regulatory partners, cyclin and Cks1.
  • CDKs regulate numerous substrates through multisite phosphorylation, a process dependent on Cks1 for secondary site modification.
  • The precise mechanisms governing the specificity and pattern of multisite phosphorylation are key to understanding cell cycle regulation.

Purpose of the Study:

  • To investigate the potential role of a newly identified phosphate-binding pocket (PP) on B-type cyclins in regulating CDK substrate phosphorylation.
  • To determine if this cyclin-associated PP influences the pattern of multisite phosphorylation, analogous to Cks1's function.
  • To explore the impact of PP mutations on cell cycle progression and substrate modification in budding yeast.

Main Methods:

  • Site-directed mutagenesis of the phosphate-binding pocket (PP) in the Clb2 cyclin of budding yeast.
  • Phenotypic analysis of PP mutants, including assessment of bud morphology and cell cycle timing (anaphase onset).
  • In vitro biochemical assays to measure multisite phosphorylation of known CDK substrates by mutated cyclin complexes.

Main Results:

  • Mutation of the PP in Clb2 resulted in altered bud morphology and a delay in anaphase onset, indicating a role in cell cycle progression.
  • The PP mutation significantly reduced multisite phosphorylation of key CDK substrates, including components of the anaphase-promoting complex/cyclosome (Cdc16, Cdc27) and the polarisome (Bud6, Spa2).
  • These findings demonstrate that the cyclin PP is essential for efficient and specific multisite phosphorylation of CDK targets.

Conclusions:

  • The phosphate-binding pocket (PP) on B-type cyclins plays a critical role in controlling the pattern of multisite phosphorylation on CDK substrates.
  • Similar to Cks1, the cyclin PP functions as a key regulator, ensuring the correct phosphorylation events occur at specific sites.
  • This mechanism contributes to the robust and precise timing of essential cell-cycle events, ensuring faithful cell division.

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