Ko Hyuk Wan, Jiang Jin, Edery Isaac
Graduate Program in Physiology and Neurobiology, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, New Jersey 08854, USA.
Nature. 2002 Dec 12;420(6916):673-8. doi: 10.1038/nature01272. Epub 2002 Nov 20.
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 Iepsilon (CKIepsilon) 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 Iepsilon. 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.
蛋白质磷酸化在以特定于一天中不同时间的方式调节生物钟蛋白的稳定性方面起着关键作用。动物生物钟的一个保守特征是周期（Per）蛋白在磷酸化和水平上呈现每日节律，这些事件对于正常的生物钟进程至关重要。酪蛋白激酶Iε（CKIε）在调节动物体内Per蛋白的磷酸化和丰度方面发挥着重要作用。这首先在果蝇中通过对双倍时间（Dbt）的表征得到证实，Dbt是脊椎动物酪蛋白激酶Iε的同源物。然而，尚不清楚Dbt如何调节Per的水平。在这里，我们使用细胞培养系统表明，Dbt促进Per的逐步磷酸化，导致过度磷酸化的异构体通过泛素 - 蛋白酶体途径快速降解。Slimb是一种在泛素 - 蛋白酶体途径中起作用的F - 盒/ WD40重复蛋白，它优先与磷酸化的Per相互作用并刺激其降解。Slimb的过表达或在时钟细胞中表达显性负性形式的Slimb会破坏果蝇的正常节律活动。我们的研究结果表明，过度磷酸化的Per通过与Slimb的相互作用被靶向到蛋白酶体。
