Casein kinase I in the mammalian circadian clock

Abstract

The circadian clock is characterized by daily fluctuations in gene expression, protein abundance, and posttranslational modification of regulatory proteins. The Drosophila PERIOD (dPER) protein is phosphorylated by the serine?threonine protein kinase, DOUBLETIME (DBT). Similarly, the murine PERIOD proteins, mPER1 and mPER2, are phosphorylated by casein kinase I epsilon (CKI), the mammalian homolog of DBT. CKIepsilon also phosphorylates and partially activates the transcription factor BMAL1. Given the variety of potential targets for CKIepsilon and other cellular kinases, the precise role of phosphorylation is likely to be a complex one. Biochemical analysis of these and other circadian regulatory proteins has proven to be a fruitful approach in determining how they function within the context of the molecular clockworks.

Figure 1

A. Purified thioredoxin-His-CKIɛ(Δ319) and MBP-mPER2(450–763). Proteins were purified as described and 5 μg analyzed by SDS-PAGE. B. Recombinant CKIɛ(Δ319) phosphorylates mPER2 to high stoichiometry. Phosphorylation of mPER2(40 pmol) by CKIɛ(Δ319) (20 pmol) was carried out at 37°C in 30 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 1 μg BSA, 250 μM ATP, 200 μCi ³²P-[ $\tilde{\gamma }$ ATP]. At each time point, the reaction was stopped by adding 1X SDS-PAGE sample buffer followed by boiling for 3 min at 95°C. The proteins in the reactions were separated on 12% SDS-PAGE. The phosphorylation of mPER2 was quantitated by Phosphor-Imager and ImageQuant software.

Figure 1

A. Purified thioredoxin-His-CKIɛ(Δ319) and MBP-mPER2(450–763). Proteins were purified as described and 5 μg analyzed by SDS-PAGE. B. Recombinant CKIɛ(Δ319) phosphorylates mPER2 to high stoichiometry. Phosphorylation of mPER2(40 pmol) by CKIɛ(Δ319) (20 pmol) was carried out at 37°C in 30 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 1 μg BSA, 250 μM ATP, 200 μCi ³²P-[ $\tilde{\gamma }$ ATP]. At each time point, the reaction was stopped by adding 1X SDS-PAGE sample buffer followed by boiling for 3 min at 95°C. The proteins in the reactions were separated on 12% SDS-PAGE. The phosphorylation of mPER2 was quantitated by Phosphor-Imager and ImageQuant software.

Figure 2

Effect of kinase inhibitors on mPER2 stability. HEK 293 cells overexpressing myc-mPER2 (450–763) were pretreated with the CKIɛ inhibitor IC261 (50 μM), MAPK inhibitor U0126 (30 μM) or vehicle for 4 hours. Next, they were treated with cycloheximide (25 μg/ml) for 20 minutes and with calyculin A 80 nM or DMSO for 0, 30 and 60 minutes. Cells lysates were analyzed by SDS-PAGE and immunoblotting with antibodies recognizing the myc epitope of PER2 and the actin proteins.