Identification of Light-Sensitive Phosphorylation Sites on PERIOD That Regulate the Pace of Circadian Rhythms in Drosophila

Abstract

The main components regulating the pace of circadian (Х24 h) clocks in animals are PERIOD (PER) proteins, transcriptional regulators that undergo daily changes in levels and nuclear accumulation by means of complex multisite phosphorylation programs. In the present study, we investigated the function of two phosphorylation sites, at Ser826 and Ser828, located in a putative nuclear localization signal (NLS) on the Drosophila melanogaster PER protein. These sites are phosphorylated by DOUBLETIME (DBT; Drosophila homolog of CK1␦/), the key circadian kinase regulating the daily changes in PER stability and phosphorylation. Mutant flies in which phosphorylation at Ser826/Ser828 is blocked manifest behavioral rhythms with periods slightly longer than 1 h and with altered temperature compensation properties. Intriguingly, although phosphorylation at these sites does not influence PER stability, timing of nuclear entry, or transcriptional autoinhibition, the phospho-occupancy at Ser826/Ser828 is rapidly stimulated by light and blocked by TIMELESS (TIM), the major photosensitive clock component in Drosophila and a crucial binding partner of PER. Our findings identify the first phosphorylation sites on core clock proteins that are acutely regulated by photic cues and suggest that some phosphosites on PER proteins can modulate the pace of downstream behavioral rhythms without altering central aspects of the clock mechanism.A wide variety of life forms exhibit circadian (ϳ24 h) rhythms in metabolism, physiology, and behavior, which are governed by cellular "clocks" based on the expression of species-or tissuespecific sets of clock genes (reviewed in reference 1). In general, clock mechanisms are biochemical oscillators built on interlocked loops of transcriptional negative feedback and protein degradation, wherein a "master" clock transcription factor drives expression of one or more key repressor proteins that, after a delay, feed back to inhibit the transcription factor until the repressor(s) declines in abundance, enabling another round of gene expression (2). This molecular logic of circadian clocks is usually referred to as transcriptional-translational feedback loops (TTFLs). Studies based on a wide range of model systems indicate that the daily changes in the levels of the key clock feedback repressor(s) are driven by complex temporal phosphorylation programs that dictate the pace of the clock (3-6). In animals, PERIOD (PER) proteins are the central components of the negative arm of the clock mechanism and behave as the primary phosphotimer regulating clock speed (3, 4). A major effect of phosphorylation on regulating the pace of the clock is via evoking temporal changes in the stability of PER proteins, which yields daily cycles in their levels that are inextricably linked to clock progression.Studies of Drosophila melanogaster have been instrumental in our understanding of clock mechanisms in general and mammalian ones in particular. The D. melanogaster intracellular clock mechanism is comprised of interlocked tran...

Phosphatase of Regenerating Liver-1 Selectively Times Circadian Behavior in Darkness via Function in PDF Neurons and Dephosphorylation of TIMELESS

“…One site (S586) is immediately adjacent to the TIM nuclear localization signal, S586, while the second site, T991, is close to PER interacting domain ( Figures 4D and S3). In the case of PER, we identified 9 phosphorylation sites ( Figure 4E), several of which have been identified as DBT phosphorylation sites by consensus prediction and/or subsequent experimental testing [69][70][71] or are sites for other known circadian kinases. 16 Of note, a number of these sites were increased in phosphorylation (i. e., S27 and S1187) in the presence of PRL-1, suggesting that PER is not a direct target of PRL-1 dephosphorylation.…”

The Function, Regulation, and Mechanism of Protein Turnover in Circadian Systems in Neurospora and Other Species

“…Dbt-dependent phosphorylation of Drosophila PER S826,S828 , two sites located in a putative nuclear localization signal (NLS), can be rapidly induced by light and blocked by TIM. However, mutation of these two sites causes only mild changes in behavioral rhythms with a slightly longer period (by 1-h), suggesting that phosphorylation of these two sites may play an unknown role rather than controlling the circadian period [ 244 ]. Notably, some of the phosphorylation events show no impact on the circadian period, likely because some phosphodegrons only function as entire phosphorylated domains rather than as partially modified amino acids [ 53 ].…”

CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms

“…35 Significant progress has been made in examining the function of site-specific PER phosphorylation, enabling in-depth mechanistic understanding of post-translational regulation of PER subcellular localization, repressor activity, and degradation to generate a 24-h rhythm. 28,29,[41][42][43][44][45] On the other hand, the relative dearth of studies that characterize site-specific functions of TIM phosphorylation 22,31,46 remains a significant obstacle to fully understand the regulation of circadian rhythms via post-translational regulation of TIM and PER-TIM complexes. In this study, we used mass spectrometry proteomics to identify phosphorylation sites of PER-bound TIM proteins purified from Drosophila heads.…”