Clock-Dependent Phosphorylation of CikA Regulates Its Activity.

Abstract

In the cyanobacterial circadian clock, a core oscillator comprising the proteins KaiA, KaiB, and KaiC keeps time based on a rhythmic phosphorylation of KaiC, and histidine protein kinases relay temporal information from the KaiABC complex to regulate gene expression. The kinases SasA and CikA engage directly with the oscillator and are responsible for modulating the phosphorylation and dephosphorylation throughout the circadian day of the response-regulator transcription factor RpaA; the phosphorylation state of RpaA in turn determines circadian gene expression. We recently showed that either CikA or SasA can drive rhythmic phosphorylation and DNA binding of RpaA in an in vitro system. However, when SasA is absent in vivo, a bioluminescence reporter gene shows a very low expression and amplitude rhythm, indicating CikA kinase activity is not sufficient to activate gene expression. We questioned why CikA cannot serve as a robust kinase for RpaA in the absence of SasA in the cell. Here, we investigated post-translational modifications of CikA and found KaiC-dependent phosphorylation sites of CikA that dramatically affect its activity. Phosphomimetic mutants of these sites showed that the phosphorylated version of CikA is not functional. Our data show that inverse correlation of KaiC levels and these inhibitory phosphorylation sites can explain the lower CikA activity in a SasA knockout background. We conclude that these phosphorylation sites act as a rheostat for CikA activity and are regulated by KaiC levels.