Dataset Information

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.

SUBMITTER: Syed S

PROVIDER: S-EPMC3149356 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein.

Syed Sheyum S Saez Lino L Young Michael W MW

The Journal of biological chemistry 20110609 31

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 rob ...[more]

PMID: 21659538

Similar Datasets

Project description:Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.

Project description: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.

Dataset Information

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein.

Publications

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets