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Abstract
The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in bothDrosophila and mammals. The Drosophila double-time gene product, a casein kinase I ɛ (CKIɛ) homolog, has been reported to interact with dPER and regulate circadian cycle length. We find that mammalian CKIɛ binds to and phosphorylates the murine circadian regulator mPER1. Unlike both dPER and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry. First, coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization. Second, coexpression of CKIɛ leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIɛ may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus.
ACKNOWLEDGMENTS
The first two authors contributed equally to this work.
We thank M. Tei, H. Okamura, M. Young, J. Takahashi, and M. Morgan for generously providing plasmids, Rebecca Shepard and Aurelia Meloni-Ehrig for assistance with immunofluorescence, Bob Schackman for oligonucleotide synthesis, and L. Ptacek, D. Ayer, B. Graves, E. Raetz, and K. Ullman for constructive criticism of the manuscript.
This work was supported by grant R01 CA71074 from the NIH to D.M.V. Oligonucleotide synthesis was supported by Cancer Center Support grant 3P30 CA42014.
ADDENDUM IN PROOF
Supporting an essential role for casein kinase Iɛ in mammalian circadian rhythm, Lowrey et al. have reported that the tauhamster locus encodes casein kinase Iɛ (P. L. Lowrey et al., Science288:483–491, 2000). Furthermore, Keesler et al. have reported in interaction of casein kinase Iɛ with human mPER1 (Keesler et al., NeuroReport 2:1–5, 2000).