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NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator.

ABSTRACT: The Drosophila circadian oscillator is comprised of transcriptional feedback loops that are activated by CLOCK (CLK) and CYCLE (CYC) and repressed by PERIOD (PER) and TIMELESS (TIM) [1]. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM [2-4]. Although kinases that control PER and TIM levels and subcellular localization have been identified [5-10], additional kinases are predicted to target PER, TIM, and/or CLK to promote time-specific transcriptional repression. We screened for kinases that alter circadian behavior via clock cell-directed RNA interference (RNAi) and identified the proline-directed kinase nemo (nmo) as a novel component of the circadian oscillator. Both nmo RNAi knockdown and a nmo hypomorphic mutant shorten circadian period, whereas nmo overexpression lengthens circadian period. CLK levels increase when nmo expression is knocked down in clock cells, whereas CLK levels decrease and PER and TIM accumulation are delayed when nmo is overexpressed in clock cells. These data suggest that nmo slows the pace of the circadian oscillator by altering CLK, PER, and TIM expression, thereby contributing to the generation of an ~24 hr circadian period.

SUBMITTER: Yu W

PROVIDER: S-EPMC3090582 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator.

Yu Wangjie W Houl Jerry H JH Hardin Paul E PE

Current biology : CB 20110421 9

The Drosophila circadian oscillator is comprised of transcriptional feedback loops that are activated by CLOCK (CLK) and CYCLE (CYC) and repressed by PERIOD (PER) and TIMELESS (TIM) [1]. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM [2-4]. Although kinases that control PER and TIM levels and subcellular localization have been identified [5-10], additional kinases are predicted to target PER, ...[more]

PMID: 21514156

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Project description:Circadian rhythms in Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and Protein Phosphatase-1 (PP1). Here, we used mass spectrometry to identify 35 "phospho-occupied" serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. We found that cell culture assays were not good predictors of protein function in flies and so we generated per transgenes carrying phosphorylation site mutations and tested for rescue of the per(01) arrhythmic phenotype. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ~ 30 hrs. Interestingly, the single S613A mutation extends the period by 2-3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. Biochemical and immunohistochemical data indicate defects in overall phosphorylation and altered timely degradation of PER carrying the double or single S613A mutation(s). The PER-T610A/S613A mutant also alters CLK phosphorylation and CLK-mediated output. Lastly, we show that a mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period.

Dataset Information

NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator.

Publications

NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator.

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