1. Graduate Program in Physiology and Neurobiology, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, New Jersey 08854, USA
2. Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, New Jersey 08854, USA
3. Center for Developmental Biology and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

Correspondence to: Isaac Edery³ Correspondence and requests for materials should be addressed to I.E. (e-mail: Email: edery@cabm.rutgers.edu).

Abstract

Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day¹. 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, 5, 6, 7}. Casein kinase I (CKI) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals⁸. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homologue of vertebrate casein kinase I^{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.