Abstract
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 per01 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.
Highlights
Circadian rhythms, exhibited by a variety of organisms ranging from bacteria to plants and humans, help to establish daily temporal organization
The clock is controlled by post-translational modifications, including phosphorylation of a core negative regulator, PERIOD (PER)
In this study, using a proteomic approach to identify phosphooccupied sites, we demonstrate that phospho-residues PERT610, and to a larger extent PER-S613, act as key regulators of the Drosophila clockwork and help to control timing of phosphorylation, overall PER stability, and circadian periodicity
Summary
Circadian rhythms, exhibited by a variety of organisms ranging from bacteria to plants and humans, help to establish daily temporal organization. These rhythms are typically generated by one or more feedback loops within core clock or ‘‘pacemaker’’ cells. Levels of PER and TIM cycle over the course of the day with peak expression occurring in the mid- to late-night. As PER expression begins to increase during the middle-late day, it is phosphorylated by DOUBLETIME (DBT) and turned over. It is only after TIM accumulates and heterodimerizes with PER at the end of the day that PER is protected from degradation. Stable nuclear expression of TIM and PER later in the night leads to a decrease in CLK/CYC heterodimer activity [1]
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