Drosophila DBT Lacking Protein Kinase Activity Produces Long-Period and Arrhythmic Circadian Behavioral and Molecular Rhythms

Abstract

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...

Drosophila and Vertebrate Casein Kinase Iδ Exhibits Evolutionary Conservation of Circadian Function

“…In vitro kinase assays with DBT S , DBT L , or C-terminally truncated forms of DBT: DBT-MYC expression was induced in stably transfected cell lines with CuSO 4 , the cell lysates were immunoprecipitated with anti-MYC antibody, the immunoprecipitates were incubated with PER expressed and purified from bacteria, and radiolabeled substrate was detected and quantified by phosphor-imager analysis of SDS-PAGE (Muskus et al 2007). Aliquots of each reaction were also analyzed by immunoblot analysis with an antibody detecting the C terminus of DBT (Figure 1) or the MYC epitope (Figure 2), and the signal for 32 P incorporation was normalized to the signal for DBT, as previously described (Muskus et al 2007).…”

“…It was first shown that PER was rhythmically phosphorylated (Edery et al 1994), and isolation and analysis of the dbt mutants in Drosophila then showed that casein kinase I (CKI) targets PER for phosphorylation and degradation Price et al 1998;Rothenfluh et al 2000a;Suri et al 2000;Muskus et al 2007). Doubletime (DBT) has also been proposed to regulate the nuclear localization (Bao et al 2001;Cyran et al 2005;Nawathean et al 2007) and the repressor capacity of PER (Nawathean and Rosbash 2004;Kim et al 2007;Nawathean et al 2007), as well as the phosphorylation of CLK protein (Kim and Edery 2006;Yu et al 2006).…”

“…The importance of dbt or vertebrate ckI protein kinases for the core time-keeping process is exemplified by mutants that can either shorten or lengthen the circadian period Lowrey et al 2000;Rothenfluh et al 2000a;Suri et al 2000;Xu et al 2005;Muskus et al 2007). Intriguingly, all of the known mutant kinases exhibit lower kinase activity on complex substrates like casein and PER in vitro (Lowrey et al 2000;Suri et al 2000;Preuss et al 2004;Xu et al 2005), with all of the long-period mutants identified in fly DBT and all but one of the short-period mutants in vertebrate CKIe/d, raising the possibility that there are differences between the mammalian and Drosophila circadian kinases and mechanisms (Xu et al 2005;Sekine et al 2008).…”

“…Surprisingly, the results suggest a high degree of evolutionary conservation for CKId and DBT and for the circadian functions affected by the short-period mutations of DBT and CKIe/d. , the catalytically inactive DBT K/R protein, or truncated forms of DBT were used to produce stably transfected S2 cell lines as previously described (Muskus et al 2007). To produce the truncated forms of DBT, PCR reactions were employed to amplify different extents of the DBT C terminus.…”

“…Digestion of the PCR product with NsiI and AgeI yielded a fragment that was ligated in place of the NsiI/AgeI fragment from pMT-DBT, yielding a plasmid that expressed an epitope-tagged truncation mutant. DBT-MYC-expressing constructs were transiently cotransfected into S2 cells with pAC-PER-HA, expression of DBT-MYC was induced with CuSO 4 , and PER and DBT levels were detected by immunoblot analysis with anti-HA and anti-MYC antibodies as previously described (Muskus et al 2007). For the experiments examining autophosphorylation of DBT (Figure 3), stably transfected lines expressing DBT WT -MYC or DBT K/R -MYC from the pMT vector were used.…”