The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator

Abstract

The CK2 kinase plays diverse roles in the Drosophila circadian clock by controlling stability of the CLOCK transcription factor itself, besides its known role in degrading CLOCK repressors.

Abstract

Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER) and TIMELESS (TIM) proteins, which inhibit CLOCK (CLK) transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic α subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory β subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of CK2α activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2.

Author Summary

The CK2 kinase is an ancient enzyme known to be at the heart of self-sustaining circadian clocks in animals, plants, and fungi. Circadian clocks are responsible for daily circadian rhythms in molecular, physiological, and behavioral processes. Their mechanism relies on transcriptional activators and repressors that constitute a feedback loop. The CLOCK (CLK) activator is required to initiate the transcription of the period ( per) and timeless ( tim) genes in the late day. The PER and TIM repressors accumulate in a delayed manner and translocate to the nucleus to repress their own gene. Degradation of these repressors allows the activator to start a new cycle. In the fruit fly Drosophila melanogaster, CK2-mediated phosphorylation of the PER and TIM repressors targets them for degradation. Here we find that the CLK activator is also regulated by CK2. In contrast to PER and TIM, CLK is stabilized by CK2α phosphorylation; we also show that PER and TIM are dispensable for this stabilization of CLK. The identification of CK2–CLK protein complexes and the in vitro phosphorylation of CLK by CK2 hint at a direct action of the kinase on the activator. Although transcription factor stabilization is generally expected to be associated with increased activity, genetic analysis indicates that CK2 represses CLK. Hence CK2 phosphorylation seems to be a key signal for the transcriptional activator complex to adopt a proper activation state at a given point of the circadian cycle.