Rapid activation of CLOCK by Ca²⁺-dependent protein kinase C mediates resetting of the mammalian circadian clock

In mammals, immediate-early transcription of the Period 1 (Per1) gene is crucial for resetting the mammalian circadian clock. Here, we show that CLOCK is a real signalling molecule that mediates the serum-evoked rapid induction of Per1 in fibroblasts through the Ca²⁺-dependent protein kinase C (PKC) pathway. Stimulation with serum rapidly induced nuclear translocation, heterodimerization and Ser/Thr phosphorylation of CLOCK just before the surge of Per1 transcription. Serum-induced CLOCK phosphorylation was abolished by treatment with PKC inhibitors but not by other kinase inhibitors. Consistently, the interaction between CLOCK and PKC was markedly increased shortly after serum shock, and the Ca²⁺-dependent PKC isoforms PKCα and PKCγ phosphorylated CLOCK in vitro. Furthermore, phorbol myristic acetate treatment triggered immediate-early transcription of Per1 and also CLOCK phosphorylation, which were blocked by a Ca²⁺-dependent PKC inhibitor. These findings indicate that CLOCK activation through the Ca²⁺-dependent PKC pathway might have a substantial role in phase resetting of the circadian clock.