Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Cell-cycle progression is regulated by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.
• The order and extent of substrate phosphorylation are influenced by Cdk activity levels and substrate-docking interactions.
• Multisite phosphorylation allows for complex regulatory mechanisms in cellular processes.

Purpose of the Study:

• To elucidate the regulatory circuit involving multisite phosphorylation of Ndd1, a transcriptional co-activator crucial for mitotic progression in budding yeast.
• To investigate the dual role of Cdk1 activity in regulating CLB2 gene expression and Ndd1 stability during mitosis.

Main Methods:

• Investigated the effect of varying Cdk1 activity on CLB2 expression and Ndd1 stability.
• Utilized biochemical assays to examine the phosphorylation status of Ndd1 by Cdk1-Clb2 complexes.
• Assessed the role of Cks1 and Clb2 binding sites in Ndd1 phosphorylation and degradation.

Main Results:

• Low Cdk1 activity promotes CLB2 expression at mitotic entry, consistent with positive feedback.
• High Cdk1 activity during mitotic arrest inhibits CLB2 expression and triggers Ndd1 degradation.
• Multisite phosphorylation of Ndd1 by high mitotic Cdk1-Clb2 activity, dependent on Cks1 and Clb2 binding sites, mediates Ndd1 degradation.

Conclusions:

• Rising Cdk1 activity orchestrates a pulse of mitotic gene expression by sequentially activating positive and negative feedback mechanisms.
• Initial Cdk1 phosphorylation primes Ndd1 for subsequent phosphorylation events that lead to degradation, fine-tuning gene expression.
• The interplay between Cdk1 activity, multisite phosphorylation, and protein degradation provides a sophisticated regulatory system for mitotic progression.