Role for Slimb in the degradation of Drosophila Period protein phosphorylated by Doubletime

Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day(1). A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels(2,3), events that are crucial for normal clock progression(4-7). Casein kinase Iepsilon (CKIepsilon) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals(8). This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homologue of vertebrate casein kinase Iepsilon(4,6). However, it is not clear how Dbt regulates the levels of Per. Here we show, using a cell culture system, that Dbt promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin-proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin-proteasome pathway(9,10) interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. Our findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb.