Bub1 autophosphorylation feeds back to regulate kinetochore docking and promote localized substrate phosphorylation

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Bub1 kinase phosphorylates histone H2A-T120, crucial for sister chromatid cohesion via shugoshin (Sgo) recruitment during mitosis.
• The precise regulation and dynamic behavior of H2A-T120 phosphorylation remain incompletely understood.

Purpose of the Study:

• To investigate the regulation and dynamics of Bub1 kinase activity and its role in H2A-T120 phosphorylation.
• To elucidate the impact of Bub1 autophosphorylation on its kinetochore localization and function in chromosome segregation.

Main Methods:

• Quantitative phosphoproteomics to identify Bub1 autophosphorylation sites.
• Site-directed mutagenesis of Bub1 autophosphorylation sites (e.g., T589A).
• Analysis of kinetochore localization, H2A-T120 phosphorylation, Sgo recruitment, and chromosome segregation in mutant cells.

Main Results:

• Bub1 undergoes extensive mitosis-specific autophosphorylation, with activation primed in interphase and completed in mitosis.
• Mutation of Bub1 autophosphorylation site T589 disrupts kinetochore turnover, leading to uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms.
• This disruption results in impaired sister chromatid resolution and increased chromosome segregation errors.
• Recruitment of the Bub1-T589A mutant to kinetochores can restore centromeric H2A-T120 phosphorylation and Sgo1 localization.

Conclusions:

• Bub1 autophosphorylation is a critical feedback mechanism controlling its activation, kinetochore residency, and localization dynamics.
• Dynamic and regulated kinetochore localization of Bub1 is essential for accurate H2A-T120 phosphorylation and proper chromosome segregation.
• These findings offer new insights into the intricate regulation of the Bub1-Bub3-BubR1 complex at kinetochores.