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Updated: Jul 3, 2026
12:26
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
A quantitative atlas of mitotic phosphorylation
Noah Dephoure1, Chunshui Zhou, Judit Villén
1Department of Cell Biology, Harvard University Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Summary
This study identified over 14,000 protein phosphorylation sites during the cell cycle, revealing new insights into cyclin-dependent kinase (CDK) regulation and potentially undiscovered kinases involved in cell division.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The eukaryotic cell division cycle is a highly regulated process crucial for cell duplication.
- Cyclin-dependent kinases (CDKs) drive the cell cycle through protein phosphorylation.
- Understanding cell cycle-regulated phosphorylation is key to deciphering cell division control.
Purpose of the Study:
- To identify proteins and phosphorylation sites regulated during the cell cycle.
- To gain deeper insights into the mechanisms of cell cycle control by phosphorylation.
- To discover novel substrates and regulators of cell cycle kinases.
Main Methods:
- Utilized stable isotope labeling and mass spectrometry for quantitative phosphoproteomics.
- Employed a two-step phosphopeptide enrichment strategy.
- Analyzed protein phosphorylation in human cells arrested in G(1) and mitotic phases.
Main Results:
- Identified over 14,000 distinct phosphorylation events, with >50% novel findings.
- Quantified relative phosphorylation changes for a majority of identified sites.
- Observed increased phosphorylation in >1,000 proteins during mitosis, including known cell cycle regulators.
- Identified conserved phosphorylation motifs ([S/T]P) suggesting CDK substrate activity.
- Discovered unique motifs in non-proline site phosphopeptides, hinting at novel kinases.
Conclusions:
- This comprehensive phosphoproteomic analysis significantly expands the known landscape of cell cycle-regulated phosphorylation.
- Findings suggest extensive CDK substrate activity and provide evidence for at least two novel mitotic kinases.
- The study offers a valuable resource for future research into cell cycle regulation and kinase discovery.
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