PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription

Abstract

Transcriptional activation by CLOCK-CYCLE (CLK-CYC) heterodimers and repression by PERIOD-TIMELESS(PER-TIM) heterodimers are essential for circadian oscillator function in Drosophila. PER-TIM was previously found to interact with CLK-CYC to repress transcription, and here we show that this interaction inhibits binding of CLK-CYC to E-box regulatory elements in vivo. Coincident with the interaction between PER-TIM and CLK-CYC is the hyperphosphorylation of CLK. This hyperphosphorylation occurs in parallel with the PER-dependent entry of DOUBLE-TIME (DBT) kinase into a complex with CLK-CYC, where DBT destabilizes both CLK and PER. Once PER and CLK are degraded, a novel hypophosphorylated form of CLK accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that PER-dependent rhythms in CLK phosphorylation control rhythms in E-box-dependent transcription and CLK stability, thus linking PER and CLK function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals.

Role of Phosphorylation in the Mammalian Circadian Clock

“…In dbt mutant flies, the stability, phosphorylation pattern, and subcellular localization of dPER is modified Price et al 1998). Subsequent studies showed an additional role for DBT in the phosphorylation of dCLOCK, thereby regulating its stability (Kim and Edery 2006;Yu et al 2006). In addition, Drosophila casein kinase 2 (dCK2) was shown to phosphorylate dPER.…”

Clk post-transcriptional control denoises circadian transcription both temporally and spatially

“…The current model states that most (if not all) of this control is through post-translational regulation such as phosphorylation, ubiquitination and other types of modifications [21][22][23][24][25] . Indeed, although Clk mRNA levels display strong transcriptional oscillations 11,12 , CLK protein levels are nearly constant though the day 26 .…”

Even a stopped clock tells the right time twice a day: circadian timekeeping in Drosophila

“…PER/TIM dimers then accumulate in the cytoplasm, and enter the nucleus ∼6 h after lights off. PER and TIM dissociate inside the nucleus, and PER represses transcription by preventing the CLK/CYC dimer from binding the per and tim promoters [28,29]. The length of the delay before nuclear entry of PER/TIM is in part dependent on the activity of the Glycogen Synthase Kinase ortholog SHAGGY (SGG) [30].…”

“…Many other factors contribute to this loop, including DOUBLE-TIME (DBT), a Casein Kinase Iɛ homologue [23], Casein Kinase II [31,32], Protein Phosphatase 2A (PP2A) [33], the F-box protein SLIMB [34,35], and the blue light photoreceptor CRYPTOCHROME (CRY), that also doubles as a transcriptional repressor in some clock cells [26,36,37]. Attention has largely focussed on how these proteins regulate the stability of PER and TIM, although DBT and PP2A also regulate CLK activity [38], with hypophosphorylated CLK associated with maximal per transcription [29,38].…”