A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBT K/R ) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBT K/R antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered "set point" for period length that is not altered by overexpression. Overexpression of the DBT K/R produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.Circadian rhythms are molecular, physiological, or behavioral processes which occur with a periodic oscillation of approximately 24 h. These rhythms can be entrained by environmental cues such as light/dark cycles, but they persist in the absence of such cues, thus demonstrating the presence of an endogenous clock (reviewed in reference 40). Drosophila melanogaster has been widely used as a model organism for a genetic analysis of circadian rhythms, and many of the gene products comprising the endogenous clock mechanism were identified in this organism, including the first discovered clock gene, period (per) (reviewed in reference 42). Molecular genetic analysis of these rhythms has elucidated a basic clock mechanism consisting of oscillating clock gene products, which regulate their own expression through positive and negative feedback loops. During the night, PER and Timeless protein (TIM) levels rise in the cytoplasm, where the proteins heterodimerize, and then translocate into the nucleus to negatively regulate the transcription of their own genes and other genes. Negative regulation is effected by interactions with the transcription factors dClock (dCLK) and Cycle (CYC), which activate transcription of the per and tim genes and indirectly repress transcription of dClk in the absence of PER and TIM. Throughout the day, TIM levels do not accumulate in the nucleus because of TIM's degradation via a light-and cryptochrome-mediated degradation pathway (reviewed in reference 42).DBT, an ortholog of mammalian casein kinase Iε and casein kinase I␦ (CKIε/␦), regulates PER cytoplasmic and nuclear accumulation by triggering PER's degradation and regulating the timing of its nuclear accumulation (3,8,9,22,23,43,46,49). DBT's activity on PER is supplemented by the activity of CKII (2, 27, 28) and SGG (31) and is antagonized by a rhythmically expressed protein phosphatase (47). DBT may regulate other aspects of PER function (35) and ot...

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...

The Drosophila double-time (dbt) gene, which encodes a protein similar to vertebrate epsilon and delta isoforms of casein kinase I, is essential for circadian rhythmicity because it regulates the phosphorylation and stability of period (per) protein. Here, the circadian phenotype of a short-period dbt mutant allele (dbt(S)) was examined. The circadian period of the dbt(S) locomotor activity rhythm varied little when tested at constant temperatures ranging from 20 to 29 degrees C. However, per(L);dbt(S) flies exhibited a lack of temperature compensation like that of the long-period mutant (per(L)) flies. Light-pulse phase-response curves were obtained for wild-type, the short-period (per(S)), and dbt(S) genotypes. For the per(S) and dbt(S) genotypes, phase changes were larger than those for wild-type flies, the transition period from delays to advances was shorter, and the light-insensitive period was shorter. Immunohistochemical analysis of per protein levels demonstrated that per protein accumulates in photoreceptor nuclei later in dbt(S) than in wild-type and per(S) flies, and that it declines to lower levels in nuclei of dbt(S) flies than in nuclei of wild-type flies. Immunoblot analysis of per protein levels demonstrated that total per protein accumulation in dbt(S) heads is neither delayed nor reduced, whereas RNase protection analysis demonstrated that per mRNA accumulates later and declines sooner in dbt(S) heads than in wild-type heads. These results suggest that dbt can regulate the feedback of per protein on its mRNA by delaying the time at which it is translocated to nuclei and altering the level of nuclear PER during the declining phase of the cycle.

SUMMARY The kinase DOUBLETIME is a master regulator of the Drosophila circadian clock, yet the mechanisms regulating its activity remain unclear. A proteomic analysis of DOUBLETIME interactors led to the identification of an unstudied protein designated CG17282. RNAi-mediated knock-down of CG17282 produced behavioral arrhythmicity and long periods, high levels of hypophosphorylated nuclear PERIOD and phosphorylated DOUBLETIME. Overexpression of DOUBLETIME in flies suppresses these phenotypes and overexpression of CG17282 in S2 cells enhances DOUBLETIME-dependent PERIOD degradation, indicating that CG17282 stimulates DOUBLETIME’s circadian function. In photoreceptors, CG17282 accumulates rhythmically in PERIOD- and DOUBLETIME-dependent cytosolic foci. Finally, structural analyses demonstrated CG17282 is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that binds DOUBLETIME and tetratricopeptide repeats that may promote assembly of larger protein complexes. We have named CG17282 Bride of Doubletime and established it as a mediator of DOUBLETIME’s effects on PERIOD, most likely in cytosolic foci that regulate PERIOD nuclear accumulation.

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase Ie/d (CKIe/d) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI shortperiod mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIe/d. Moreover, expression of either Drosophila DBT or the vertebrate CKId kinase carrying the Drosophila dbt S or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKId carrying the dbt L mutation does not lengthen circadian rhythms, while Drosophila DBT L does. Different effects of the dbt S and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKId and of the functions affected by their period-shortening mutations.

While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.

No abstract

Doubletime (DBT) has an essential circadian role in Drosophila melanogaster because it phosphorylates Period (PER). In order to determine if DBT antagonism can produce distinct effects in the cytosol and nucleus, forms of a dominant negative DBTK/R with these two alternative localizations were produced. DBT has a putative nuclear localization signal (NLS), and mutation of this signal confers cytosolic localization of DBT in the lateral neurons of Drosophila clock cells in the brain. By contrast, addition of a strong NLS domain (e.g. SV40 NLS) to DBT’s C terminus leads to more nuclear localization. Expression of DBTK/R with the mutated NLS (DBTK/R NLS−) using a timGAL4 driver does not alter the circadian period of locomotor activity, and the daily oscillations of PER detected by immunoblot and immunofluorescence persist, like those of wild type flies. By contrast, expression of DBTK/R with the strong NLS (DBTK/R stNLS) using the timGAL4 driver lengthens period more strongly than DBTK/R, with damped oscillations of PER phosphorylation and localization. Both DBTK/R and DBTWT without the NLS fail to interact with Bride of Doubletime protein (BDBT), which is related to FK506-binding proteins and shown to interact with DBT to enhance its circadian function. This result suggests that the DBTK/R NLS− has lost its dominant negative property because it does not form normal clock protein complexes. DBTWT proteins with the same changes (NLS- and stNLS) also produce equivalent changes in localization that do not produce opposite period phenotypes. Additionally, a DBTK/R protein with both the stNLS and NLS− mutation does not affect circadian period although it is nuclear, demonstrating that the lack of a dominant negative for the DBTK/R NLS− is not due to failure to localize to nuclei. Finally, bdbt RNAi increases the cytosolic localization of DBTK/R but not of DBTWT, suggesting a role for BDBT in DBT-kinase dependent nuclear localization of DBT.