Deciphering the Interplay among Multisite Phosphorylation, Interaction Dynamics, and Conformational Transitions in a...

Area of Science:

• Molecular Biology
• Biochemistry
• Proteomics

Background:

• Multisite phosphorylation regulates protein function but is challenging to analyze biochemically.
• Understanding phosphorylation dynamics is key for cell cycle regulation, especially after DNA damage.
• Polo-like kinase 1 (Plk1) activation by Aurora kinase A (Aur-A) and Bora is vital for mitotic entry.

Purpose of the Study:

• To characterize the multisite phosphorylation of Bora and its impact on Plk1 activation.
• To elucidate the sequential order and mechanistic consequences of Bora phosphorylation.
• To establish a mass spectrometry-based strategy for analyzing complex phosphorylation events.

Main Methods:

• Integration of native MS, cross-linking MS, bottom-up and top-down proteomics.
• Ion mobility spectrometry-MS to analyze structural changes.
• Characterization of the Plk1, Aur-A, and Bora protein complex.

Main Results:

• Aur-A/Bora and Plk1/Bora heterodimers form during Plk1 activation.
• Plk1/Bora interaction depends on extensive Bora multisite phosphorylation.
• Bora phosphorylation occurs sequentially, involving up to 16 sites, and induces structural changes.

Conclusions:

• A sequential, ordered mechanism for multisite Bora phosphorylation was revealed.
• Multisite phosphorylation drives structural rearrangements critical for Plk1/Bora complex formation.
• The developed MS strategy is effective for dissecting complex multisite phosphorylation events.