Large-scale phosphomimetic screening identifies phospho-modulated motif-based protein interactions.

Area of Science:

• Molecular Biology
• Post-Translational Modifications
• Proteomics

Background:

• Phosphorylation regulates protein function by altering protein-protein interactions.
• Identifying functionally characterized phosphosites remains a significant challenge.
• Understanding phospho-modulation is key to deciphering cellular regulation.

Purpose of the Study:

• To develop a method for discovering phosphosites that modulate short linear motif-based interactions.
• To screen a comprehensive library of phosphosites for their role in protein interactions.
• To functionally and structurally characterize novel phospho-modulated interactions.

Main Methods:

• Generation of a phosphomimetic proteomic peptide-phage display library covering ~13,500 human phosphosites.
• Screening of the library against 71 protein domains to identify modulating phosphosites.
• Affinity measurements and structural characterization of key interactions, including the clathrin-HURP complex.

Main Results:

• Identified 248 phosphosites that modulate motif-mediated interactions.
• Confirmed phospho-modulation for 14 out of 18 tested interactions.
• Demonstrated the essentiality of phospho-dependency in the mitotic function of HURP via its interaction with clathrin.

Conclusions:

• The phosphomimetic peptide-phage display (ProP-PD) is a powerful tool for discovering novel phospho-modulated interactions.
• These findings highlight the importance of phosphorylation in regulating protein interactions and cellular processes.
• The study provides molecular insights into the phospho-dependency of the clathrin-HURP interaction during mitosis.