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Updated: Apr 26, 2026
12:26
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
18.3K
Method for identifying phosphorylated substrates of specific cyclin/cyclin-dependent kinase complexes
Yinyin Li1, Frederick R Cross2, Brian T Chait3
1Laboratory of Mass Spectrometry and Gaseous Ion Chemistry and.
Summary
Researchers developed a novel method to identify cyclin/cyclin-dependent kinase (CDK) substrates and their phosphorylation sites. This technique revealed that Clb5/Cdc28 inactivates Cdc14 phosphatase, regulating cell cycle balance.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cell cycle progression in eukaryotes relies on precise regulation by cyclin/cyclin-dependent kinase (CDK) complexes.
- Dissecting CDK substrates specific to cell cycle stages is crucial for understanding cell cycle control.
Purpose of the Study:
- To develop and apply a mass spectrometry-based method for identifying cyclin/CDK substrates and their phosphorylation sites.
- To investigate substrates of Clb5/Cdc28 and Cln2/Cdc28 during S phase in Saccharomyces cerevisiae.
Main Methods:
- A novel in vivo/in vitro method combining native kinase-substrate complex isolation with on-bead phosphorylation using heavy-labeled ATP (ATP-γ-(18)O4).
- Mass spectrometry was employed for high-throughput identification of substrates and phosphorylation sites.
Main Results:
- The method successfully identified substrates for Clb5/Cdc28 and Cln2/Cdc28 during S phase.
- A key finding was the specific phosphorylation of Cdc14 at S429 by Clb5/Cdc28.
- This phosphorylation significantly reduces Cdc14 activity, impacting the balance between CDK and phosphatase activity.
Conclusions:
- The developed method is effective for identifying cyclin/CDK substrates and phosphorylation sites.
- Clb5/Cdc28-mediated phosphorylation of Cdc14 plays a critical role in regulating cell cycle progression by modulating phosphatase activity.
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