Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT), a Drosophila ortholog of human casein kinase I (CKI)ɛ/δ. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER). DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The perS mutation, which is associated with short-period (19-h) circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function.
Most proteins involved in circadian transcriptional feedback loops undergo reversible chemical modifications (called phosphorylation) that regulate their activity in a time-of-day–dependent manner. Doubletime (DBT), a Drosophila kinase, phosphorylates the circadian transcriptional repressor PERIOD (PER). Mutations of dbt shorten or lengthen the period of circadian behavioral rhythms, or abolish the rhythms altogether in flies. A mutation of the human ortholog of dbt, casein kinase I (CKI)δ, has been associated with certain forms of a heritable sleep disorder. The disorder may reflect altered activity of a human PER protein, as the syndrome can also be caused by mutation of a CKIɛ/δ phosphorylation site within PER2. In this study, we locate DBT-directed phosphorylation sites in the Drosophila PER protein, including a DBT target region of PER that was previously shown to regulate DBT activity. Two PER domains within this region appear to serve as alternative targets for DBT. Phosphorylation of the upstream domain seems to suppress phosphorylation elsewhere in the region, producing a stable PER protein with little activity as a transcriptional repressor. However, when phosphorylation of the upstream domain is blocked, downstream DBT targets appear to be phosphorylated, producing a highly active, but short-lived repressor. Our results suggest that ordered patterns of DBT-directed phosphorylation contribute to the timing of PER's function and disappearance, and thus influence the pace of the circadian clock.
Two phosphorylation domains inDrosophila PERIOD protein interact in a switch-like fashion with each other and the kinase DOUBLETIME to regulate PER's stability and activity as a transcriptional repressor in the circadian transcriptional feedback loop.