The Double-Time Protein Kinase Regulates the Subcellular Localization of theDrosophilaClock Protein Period

Abstract

The Period (PER), Timeless (TIM), and Double-Time (DBT) proteins are essential components of one feedback loop in the Drosophila circadian molecular clock. PER and TIM physically interact. Coexpression of PER and TIM promotes their nuclear accumulation and influences the activity of DBT: although DBT phosphorylates and destabilizes PER, this is suppressed by TIM. Experiments using Drosophila cells in culture have indicated that PER can translocate to the nucleus without TIM and will repress transcription in a DBT-potentiated manner. In this study, we examined the control of PER subcellular localization in Drosophila clock cells in vivo. We found that PER can translocate to the nucleus in tim 01 null mutants but only if DBT kinase activity is inhibited. We also found that nuclear PER is a potent transcriptional repressor in dbt mutants in vivo without TIM. Thus, in vivo, DBT regulates PER subcellular localization, in addition to its previously documented role as a mediator of PER stability. However, DBT does not seem essential for transcriptional repression by PER. It was reported previously that overexpression of a second kinase, Shaggy (SGG)/Glycogen Synthase Kinase 3, accelerates PER nuclear accumulation. Here, we show that these effects of SGG on PER nuclear accumulation require TIM. We propose a revised clock model that incorporates this tight kinase regulation of PER and TIM nuclear entry.Key words: circadian; Period; Double-Time; Timeless; nuclear entry; Drosophila IntroductionRhythms of gene expression and protein levels lie at the core of the molecular clocks that regulate circadian (ϳ24 h) rhythms of behavior and physiology. The best-studied circadian feedback loop involves the Drosophila clock genes period (per) and timeless (tim). Many components of this negative feedback loop play similar roles in the mammalian clock (Panda et al., 2002;Stanewsky, 2003).In Drosophila, per and tim transcription is activated by Clock (CLK)/Cycle (CYC) heterodimers binding to E-boxes in the per and tim promoters. PER and TIM proteins heterodimerize in the cytoplasm, and, until recently (see below), it was believed that they enter the nucleus together. PER is more stable than TIM in the nucleus and represses additional per and tim transcription by directly inhibiting CLK/CYC.The PER/TIM loop is characterized by delays that separate transcription from repression, important for rhythmic, as opposed to continuous, negative feedback. Peak per RNA and protein levels are separated by ϳ6 h at least partly by Double-Time (DBT)/Casein Kinase 1⑀ (CK1⑀), a protein kinase that destabilizes cytoplasmic PER while TIM levels are low Suri et al., 2000). Thus, PER levels are constitutively low in tim 01 mutants (Price et al., 1995). DBT stably associates with PER (Kloss et al., 2001) and phosphorylates PER Ko et al., 2002) (S. Kivimae and M. W. Young, unpublished data), which leads to PER ubiquitination and degradation (Grima et al., 2002;Ko et al., 2002). These in vivo findings led to the model that rising TIM levels allow the formation...

A proposal for robust temperature compensation of circadian rhythms

“…The per L allele encodes a mutant protein PER L with a single amino acid substitution, resulting in long-period rhythms (Ϸ28 h) (32) that are not temperature compensated (14). In mutant cells, nuclear translocation of PER L is delayed (31,33 (14,(34)(35)(36), we favor regulated nuclear import and/or export as a likely candidate for our resetting variable.…”

Role of Phosphorylation in the Mammalian Circadian Clock

“…For example, in dbt and per mutant flies, besides the accumulation profile, the nuclear import and nuclear clearance patterns of dPER are altered. Furthermore, Cyran et al (2005) showed that phosphorylation by DBT retains PER in the cytoplasm, whereas hypophosphorylated dPER is able to enter the nucleus in tim 01 ; dbt P double-mutant larvae and tim 01 ; dbt ar double-mutant flies, respectively. Thus, proper dPER phosphorylation by DBT is crucial for correct timing of dPER nuclear entry and export.…”

The Genetics of Circadian Rhythms

“…(Table 1) In both flies and mammals a key protein kinase is DOUBLETIME (DBT)/CASEIN KINASE I delta and epsilon (CK1δ/ε). In Drosophila, DBT binds to and phosphorylates PER, regulating its subcellular localization and signaling its rapid degradation by the proteasome (25,26). The mammalian orthologs of DBT are CK1δ/ε, which mediate phosphorylation and degradation of mPER2 as well as facilitating its entry into the nucleus (27).…”