Temperature-insensitive reaction in the mammalian circadian clock

Abstract

The striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. A chemical-biological approach with 1260 pharmacologically active compounds identified “potent” compounds that markedly (>10 SD) lengthened the period. Most inhibited casein kinase Ie (CKIe) phosphorylation of the PER2 protein. By examining period extending effect and PER2 degradation inhibition of the 10 potent compounds, we verified the CKIe-dependent phosphorylation as a period-determining process. Moreover, the CKIe-dependent phosphorylation was temperature-insensitive in living clock cells. This temperature-insensitivity was preserved in the CKIe-dependent phosphorylation of a synthetic peptide in vitro. In addition, overexpression of CKIe restored temperature-insensitivity of degradation of LUC::PER2 protein in NIH3T3 cells. These results indicated that CKIe-dependent phosphorylation is a temperature-insensitive period-determining process in the mammalian circadian clock.