Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day. A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels, events that are crucial for normal clock progression. Casein kinase Iε (CKIε) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homologue of vertebrate casein kinase Iε. 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 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.
|Number of pages||6|
|Publication status||Published - 2002 Dec 12|
Bibliographical noteFunding Information:
Acknowledgements We thank J. Kong for technical assistance in isolating mec-6 cDNAs. This work was supported by the National Institutes of Health (M.C.), a Human Frontiers Science Program postdoctoral fellowship (D.S.C.), and a postdoctoral fellowship from the National Institute of Deafness and other Communication Disorders (M.B.G.).
Acknowledgements We thank S. Kay for the pAct-per and pAct-tim constructs, A. Seghal for the pCasper-hs-Ub/HA construct, J. Hall for the tim-gal4-27 and tim-gal4-62 lines, and M. Young for the tim-(UAS)-gal4 line. The work was supported by the Searle Scholar Program and the UTSW Endowed Scholar Program to J.J., and by a grant from the National Institutes of Health to I.E.
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