Abstract
Circadian clock mechanisms have been extensively investigated but the main rate-limiting step that determines circadian period remains unclear. Formation of a stable complex between clock proteins and CK1 is a conserved feature in eukaryotic circadian mechanisms. Here we show that the FRQ-CK1 interaction, but not FRQ stability, correlates with circadian period in Neurospora circadian clock mutants. Mutations that specifically affect the FRQ-CK1 interaction lead to severe alterations in circadian period. The FRQ-CK1 interaction has two roles in the circadian negative feedback loop. First, it determines the FRQ phosphorylation profile, which regulates FRQ stability and also feeds back to either promote or reduce the interaction itself. Second, it determines the efficiency of circadian negative feedback process by mediating FRQ-dependent WC phosphorylation. Our conclusions are further supported by mathematical modeling and in silico experiments. Together, these results suggest that the FRQ-CK1 interaction is a major rate-limiting step in circadian period determination.
Highlights
Circadian clocks control daily rhythms of molecular and physiological activities to allow organisms to adapt to daily environmental changes
Because CK1a forms a stable complex with FRQ and it phosphorylates FRQ but is recruited by FRQ to mediate WHITE COLLAR (WC) phosphorylation to close the circadian negative feedback loop[13,44,48], we examined the FRQ-CK1a interaction in these strains
The reduced FRQ-CK1a interaction but not FRQ stability can potentially explain the long period phenotypes of both mutants. These results suggest that the FRQ phosphorylation events impaired in these two mutants promote the FRQ-CK1a interaction
Summary
Circadian clocks control daily rhythms of molecular and physiological activities to allow organisms to adapt to daily environmental changes. Mutations of clock protein phosphorylation sites or of kinases alter protein stability and result in changes in period length[8,9,10,11,12,13,14,15,16,17], which led to the proposal that degradation rates of the negative elements is the major factor that determines the period length. It was shown that in Neurospora mutants with a defective FREQUENCY (FRQ) degradation pathway, FRQ degradation rate and circadian period length can be uncoupled[7,23] This result suggested that FRQ phosphorylation but not protein stability plays an important role in period determination. By using a combination of genetic, biochemical, mathematical modeling and in silico experiments, our results support that the formation of the FRQ-CK1a complex is the major rate-limiting process in circadian period determination
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