Clock-Dependent Phosphorylation of CikA Regulates Its Activity

Area of Science:

• * Molecular biology
• * Chronobiology
• * Biochemistry

Background:

• * The cyanobacterial circadian clock uses the KaiABC oscillator and histidine kinases to regulate gene expression.
• * SasA and CikA are key kinases that modulate RpaA phosphorylation, controlling circadian gene expression.
• * In vitro studies show CikA can phosphorylate RpaA, but in vivo, CikA alone is insufficient for robust gene expression rhythms when SasA is absent.

Purpose of the Study:

• * To investigate why CikA kinase activity is insufficient in vivo without SasA.
• * To identify post-translational modifications of CikA that affect its function.
• * To elucidate the regulatory mechanism of CikA activity within the cyanobacterial circadian clock.

Main Methods:

• * Investigated post-translational modifications of CikA.
• * Utilized phosphomimetic mutants to assess the impact of specific phosphorylation sites.
• * Analyzed the correlation between KaiC levels and CikA phosphorylation.

Main Results:

• * Identified KaiC-dependent phosphorylation sites on CikA that significantly impact its activity.
• * Demonstrated that phosphomimetic mutants of these sites render CikA non-functional.
• * Found an inverse correlation between KaiC levels and these inhibitory phosphorylation sites, explaining reduced CikA activity in SasA knockout backgrounds.

Conclusions:

• * CikA activity is regulated by KaiC-dependent phosphorylation sites that act as an inhibitory rheostat.
• * These phosphorylation sites are modulated by KaiC levels, providing a mechanism for robust circadian output.
• * The interplay between KaiC levels and CikA phosphorylation is crucial for proper functioning of the cyanobacterial circadian clock.