Drosophila doubletime Mutations Which either Shorten or Lengthen the Period of Circadian Rhythms Decrease the Protein Kinase Activity of Casein Kinase I

Abstract

In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbt S ) and long-period (dbt L ) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbt S and dbt L mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBT S , and DBT L proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbt S and dbt L mutations reduced the CKI-7-sensitive kinase activity of an orthologous Xenopus laevis CKI␦ expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbt S and dbt L mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes.Many daily biochemical, physiological, and behavioral processes are termed circadian rhythms because they are temporally regulated by an endogenous circadian clock. While these endogenous clocks are usually synchronized by the environmental light-dark or temperature cycle, in the absence of environmental cues their oscillations persist with a period of approximately 24 h (reviewed in reference 48). A genetic analysis in Drosophila melanogaster, as well as in other model organisms, has revealed much about the molecular components and mechanism of the circadian clock (reviewed in reference 74). Recently, it has become clear that the mammalian circadian clock mechanism is quite similar to the Drosophila mechanism (reviewed in reference 4).Central to the molecular mechanism of Drosophila are the oscillations of the per, tim, and dClk gene products, which drive transcriptional-translational feedback loops (3,5,16,24,27,37,59,60,75; reviewed in reference 72). PER and TIM proteins accumulate during the night, become phosphorylated, dimerize, and enter the nucleus (14,18,22,29,47,50,56,61,70,76,77), where they negatively regulate transcription of their own mRNAs (11,16,17,26,27,43,63,71,75) and positively regulate transcription of the dClk mRNA (5, 24). Both the negative and positive feedback regulation are thought to result from direct protein-protein interactions of PER and/or TIM with a CLK/CYC transcription factor (11, 37, 71) which, in the absence of PER and TIM, activates transcription of per, tim, and vrille (3,7,11,16,17,24,26,37,43,54,71) and represses the transcription of dClk (15,23,24). Entrainment, or synchronization of the clock to light-dark cycles, is conferred in part by a crypt...

An opposite role for tau in circadian rhythms revealed by mathematical modeling

“…We conclude that the tau mutation causes a site-specific increase in PER protein phosphorylation at sites that stimulate degradation. Mutations in two other members of the CKI family also cause short circadian periods and a decrease in vitro CKI kinase activity (12,35,36). Our detailed quantitative model suggests, however, that all short period mutations in CKI family members will produce a gain of function on circadian substrates.…”

“…CKI and the closely related CKI␦ are widely expressed serine-threonine protein kinases implicated in development, circadian rhythms, and DNA metabolism (11). When tested in vitro on multiple substrates, CKI tau was shown to have a much reduced overall catalytic activity (10,12,13). This partial loss-of-function mutation and its phenotype have been difficult to reconcile with our current understanding of the molecular feedback loop that governs timing in mammalian cells (13) and recent empirical observations on clock function (14-16).…”

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

“…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 hyperphosphorylation of PER (6,11) in the presence of DBT S implying that the mutant is a hypermorph.…”

“…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 is expressed in Drosophila embryonic Schneider 2 (S2) cells, but at levels that do not produce detectable phosphorylation of exogenous PER by gel shift (6,14). However, overexpression of the kinase in these cells using a constitutive promoter has been shown to qualitatively recapitulate phosphorylation pattern of overexpressed PER (6,9). The PER phosphorylation experiments were done on cell ensembles, which expressed proteins with slowly activated promoters over several hours or even days.…”