Cell-based inhibitor screening identifies multiple protein kinases important for circadian clock oscillations

Molecular oscillation of the circadian clock is based on E-box-mediated transcriptional feedback loop formed with clock genes and their encoding products, clock proteins. The clock proteins are regulated by post-translational modifications such as phosphorylation. We investigated the effects of a series of kinase inhibitors on gene expression rhythms in Rat-1 fibroblasts. The period of the cellular circadian rhythm in culture was lengthened by treatment with SB203580 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), IC261 (CKI inhibitor) and Roscovitine (CDK inhibitor). On the other hand, the period was shortened by SB216763 (GSK-3 inhibitor) or KN93 (CaMKII inhibitor) treatment. Application of 20 μM KN93 completely abolished the rhythmic gene expression. The activity of CaMKII exhibited circadian variation in a phase close to the E-box-mediated transcriptional rhythms. In vitro kinase assay revealed that CaMKII directly phosphorylates N-terminal and Ser/Pro-rich domains of CLOCK, an activator of E-box-mediated transcription. These results indicate a phosphorylation-dependent tuning of the period length by a regulatory network of multiple kinases and reveal an essential role of CaMKII in the cellular oscillation mechanism.