Decoupling PERIOD phosphorylation, stability and its rhythmic expression from mammalian circadian clock function

PERIOD proteins, the core components of the mammalian circadian negative feedback loop, exhibit robust circadian rhythms of abundances and phosphorylation profiles, which are thought be essential for clock functions. PERIOD stability was proposed to be a major circadian period length determinant. Clock proteins and CK1 form stable complexes in eukaryotic mechanisms. Here we show that mutations of PERIOD residues essential for PER-CK1 interaction in cells and in mPer2 knock-in mice abolished PERIOD2 phosphorylation and CLOCK hyperphosphorylation, resulting in PERIOD stabilization, loss of PERIOD2 abundance rhythm and impaired negative feedback process. Surprisingly, the mutant mice exhibit short period locomotor and molecular circadian rhythms, indicating that circadian clock can function independent of PERIOD phosphorylation and its abundance rhythm and period length can be uncoupled from PERIOD stability. Our results establish two circadian negative feedback mechanisms critical for clock function.