Kinetics of Doubletime Kinase-dependent Degradation of the Drosophila Period Protein

Abstract

Robust circadian oscillations of the proteins PERIOD (PER)and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT S ), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT S in cell culture resembles that with wild-type DBT and posits that, in flies DBT S likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation. During a 24-h cycle, levels of PERIOD (PER)2 and TIMELESS (TIM) proteins peak in the late night and begin to diminish thereafter. Genetic and biochemical studies suggest that the kinase Doubletime (DBT), an ortholog of the vertebrate casein kinase 1⑀, modulates phosphorylation and the early morning degradation of PER in core clock neurons (1-4). Degradation of PER occurs through the ubiquitin-proteasome pathway, where the DBT-phosphorylated PER binds the F-box protein SLIMB and is targeted for degradation (5-7). In vivo, DBT physically interacts with PER and is found to be associated with the PER-TIM complex (8) with TIM stabilizing PER by suppressing PER phosphorylation (1, 6).Several mutations of the dbt locus have been isolated and the short-period (dbt S ) and the long-period (dbt L ) alleles are among the best characterized dbt mutants in the literature (1-3, 6, 9). dbt L is a Met-80 3 Ile mutation that causes period lengthening of PER and TIM oscillations and animal behavioral activity, to ϳ27 h. Alterations in PER cyclic profile suggest that the DBT L mutation extends the animal rhythms by reducing the rate of PER phosphorylation. Indeed, in vitro gel shift and phosphorylation experiments indicate DBT L has decreased kinase activity compared with DBT (6, 9, 10), which would likely lead to slower degradation of PER in clock neurons. However, the predicted reduction in PER turnover rate due to the long-mutation on DBT has not been tested. dbt S has a Pro-47 3 Ser mutation that results in shortened PER and TIM oscillation period and fly locomotor activity interval of ϳ18 -20 h. From in vivo studies there is no conclusive evidence as to the mechanism via which the mutation affects period length. In vitro, some studies show a ϳ10% decrease in kinase activity for DBT S when compared with DBT (9, 10), suggesting that the mutant is a hypomorph. In cell culture, gel shift assays report hype...

Casein Kinase 1 Promotes Synchrony of the Circadian Clock Network

“…In Drosophila, substrate recognition of DBT seems to be temporally and spatially regulated (46,47). A reduced rate of PER degradation can explain the behavioral phenotypes of dbt AR and dbt L (48) but not of dbt S , although all produce DBT proteins that have overall reduced kinase activity (4,17,19,22,23,48). In addition, DBT may play a noncatalytic role in influencing CLKdependent transcription (18).…”

Protein sequestration versus Hill‐type repression in circadian clock models

“…fragmented free-running behavior [103,104], supporting the critical role of Rev-erbs in generating robust rhythms. Furthermore, just as the slower additional NFL is more effective in PS models [59], the half-lives of activators are also considerably longer than those of repressors in mammals [121][122][123][124][125], Drosophila [126,127] and Neurospora [76]. Tight regulation of activator level via an additional NFL (Fig.…”

Coordination between Differentially Regulated Circadian Clocks Generates Rhythmic Behavior

“…If DBT S is not impaired in PER degradation, but can still impose a short behavioral rhythm on the fly, then DBT must play an additional role in regulating behavioral rhythmicity. One possibility is that DBT S leads to early termination of PER transcription inhibition (Bao et al 2001;Syed et al 2011). The presence of DBT, but not its kinase activity, is required to hyperphosphorylate and inactivate CLK to release it from DNA (Yu et al 2009;Menet et al 2010), which may be how dbt S flies exhibit a short behavioral rhythm.…”

“…Because DBT cannot phosphorylate CLK in vitro or interact with it directly, DBT likely mediates its effect on CLK through PER (Kim and Edery 2006;Yu et al 2006;Kivimäe et al 2008;Menet et al 2010) by either being recruited to phosphorylate CLK, or through phosphorylation of PER to influence its repressor function. Thus, while mutants like DBT L are defective in triggering timely degradation of PER protein (Syed et al 2011), DBT S may be defective in regulating the activity of CLK protein.…”

“…Of the DBT mutants, surprisingly, both long and short forms exhibit reduced kinase activity (Preuss et al 2004;Kivimäe et al 2008), suggesting that DBT may serve two opposing roles in regulating rhythmicity. Indeed while DBT L and a DBT variant lacking ATPase activity (DBT K38R ) change the kinetics of PER degradation that reflect changes in behavioral rhythmicity (i.e., delayed PER degradation, longer behavioral periodicity), DBT S does not appear to alter the kinetics of PER degradation at all (Preuss et al 2004;Syed et al 2011). If DBT S is not impaired in PER degradation, but can still impose a short behavioral rhythm on the fly, then DBT must play an additional role in regulating behavioral rhythmicity.…”