Casein Kinase 1 Proteomics Reveal Prohibitin 2 Function in Molecular Clock

Abstract

Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite) with external cues (e.g., daylight and food). In vertebrates, both casein kinase 1 delta and epsilon (CK1(\delta) and CK1(\epsilon)) regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1(\delta) and CK1(\epsilon) mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2) as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1(\epsilon) expression increases PHB2 protein levels and, unexpectedly, knocking down CK1(\delta) decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1(\delta) and CK1(\epsilon) differentially regulate the expression of PHB2.