Identification of RACK1 and Protein Kinase Cα as Integral Components of the Mammalian Circadian Clock

At the core of the mammalian circadian clock is a negative feedback loop in which the dimeric transcription factor CLOCK-BMAL1 drives processes that in turn suppress its transcriptional activity. To gain insight into the mechanisms of circadian feedback, we analyzed mouse protein complexes containing BMAL1. Receptor for activated C kinase-1 (RACK1) and protein kinase C-alpha (PKC alpha) were recruited in a circadian manner into a nuclear BMAL1 complex during the negative feedback phase of the cycle. Overexpression of RACK1 and PKC alpha suppressed CLOCK-BMAL1 transcriptional activity, and RACK1 stimulated phosphorylation of BMAL1 by PKC alpha in vitro. Depletion of endogenous RACK1 or PKC alpha from fibroblasts shortened the circadian period, demonstrating that both molecules function in the clock oscillatory mechanism. Thus, the classical PKC signaling pathway is not limited to relaying external stimuli but is rhythmically activated by internal processes, forming an integral part of the circadian feedback loop.