Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Cell-cycle progression is regulated by cyclin-dependent kinases (CDKs) activated by cyclins.
• The molecular mechanisms underlying distinct cyclin functions in cell-cycle control are not fully understood.

Purpose of the Study:

• To investigate the substrate specificity of budding yeast S-phase cyclin Clb5 and M-phase cyclin Clb2 in complex with Cdk1 (Cdc28).
• To elucidate the molecular basis for differential cyclin specificity in CDK-mediated phosphorylation.

Main Methods:

• Comparative analysis of Cdk1 phosphorylation on 150 substrates using Clb5 and Clb2.
• Identification of substrate motifs and interaction interfaces responsible for cyclin specificity.
• In vivo validation using cyclin replacement and substrate motif mutagenesis.

Main Results:

• Approximately 24% of tested Cdk1 substrates were preferentially phosphorylated by Clb5-Cdk1 compared to Clb2-Cdk1.
• Key S-phase proteins like Sld2, Cdc6, Orc6, Mcm3, and Cdh1 were identified as Clb5-specific targets.
• Clb5 specificity was attributed to a hydrophobic patch interaction with substrate RXL/Cy motifs, crucial for in vivo phosphorylation.
• Clb2-Cdk1 exhibited higher intrinsic kinase activity, phosphorylating a broader range of mitotic substrates without high specificity.

Conclusions:

• Budding yeast cyclins Clb5 and Clb2 employ distinct mechanisms to achieve substrate specificity.
• Clb5 utilizes a specific interaction to target S-phase proteins, while Clb2-Cdk1's broad activity supports M-phase progression.