title: TIMELESS is an important mediator of CK2 effects on circadian clock function in vivo.
abstract: Circadian oscillations in clock components are central to generation of self-sustained 24-h periodicity. In the Drosophila molecular clock, accumulation, phosphorylation, and degradation of PERIOD (PER) and TIMELESS (TIM) proteins govern period length. Yet little is known about the kinases that phosphorylate TIM in vivo. It has been shown previously that the protein kinase CK2 phosphorylates TIM in vitro. Here, we identify a role for CK2 in TIM regulation in vivo. Induction of a dominant-negative CK2alpha, CK2alpha(Tik) (Tik), increases TIM protein and tim transcript levels, reduces oscillation amplitude, and results in persistent cytoplasmic TIM localization. Exposure to light and subsequent TIM degradation results in an increase in the fraction of the transcriptional repressor PER that is nuclear and suppression of per and tim RNA levels. TIM protein, but not tim transcript, levels are elevated in Tik mutants in a per(01) background. In contrast, Tik effects on PER are undetectable in a tim(01) background, suggesting that TIM is required for CK2 effects on PER. To identify potential CK2 target sites, we assayed TIM phosphorylation rhythms in a deletion mutant that removes a conserved serine-rich domain and found that TIM protein does not show robust rhythmic changes in mobility by Western blotting, a hallmark of rhythmic phosphorylation. The period lengthening effects in Tik heterozygotes are reduced in a tim(UL) mutant that disrupts a putative CK2 phosphorylation site. Together, these data indicate that TIM is an important mediator of CK2 effects on circadian rhythms.
authors.affiliation: Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Rose-Anne
authors.initials: RA
authors.lastname: Meissner
authors.forename: Valerie L
authors.initials: VL
authors.lastname: Kilman
authors.forename: Jui-Ming
authors.initials: JM
authors.lastname: Lin
authors.forename: Ravi
authors.initials: R
authors.lastname: Allada
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism*; D002940:Circadian Rhythm / Q000235:genetics / Q000502:physiology*; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D009154:Mutation / Q000502:physiology; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D056950:Period Circadian Proteins; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013997:Time Factors
chemical_list: D029721:Drosophila Proteins; D009687:Nuclear Proteins; C539040:PER protein, Drosophila; D056950:Period Circadian Proteins; C096445:tim protein, Drosophila; D012694:Serine; D047390:Casein Kinase II