Identification of Light-Sensitive Phosphorylation Sites on PERIOD That Regulate the Pace of Circadian Rhythms in Drosophila Evrim Yildirim, a * Joanna C. Chiu, b * Isaac Edery c Graduate Program in Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey, USA a ; Rutgers University, Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey, USA b ; Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey, USA c The main components regulating the pace of circadian (24 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 pro- grams. In the present study, we investigated the function of two phosphorylation sites, at Ser826 and Ser828, located in a puta- tive 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 peri- ods 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 (24 h) rhythms in metabolism, physiology, and behavior, which are governed by cellular “clocks” based on the expression of species- or tissue- specific 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 degrada- tion, wherein a “master” clock transcription factor drives expres- sion of one or more key repressor proteins that, after a delay, feed back to inhibit the transcription factor until the repressor(s) de- clines 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 dic- tate the pace of the clock (3–6). In animals, PERIOD (PER) pro- teins 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 stabil- ity 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 mamma- lian ones in particular. The D. melanogaster intracellular clock mechanism is comprised of interlocked transcriptional feedback loops with overlaying posttranslational regulatory circuits (re- viewed in reference 7). Prominent players in the first or major TTFL are PER (referred to here as Drosophila PER [dPER]), TIMELESS (TIM), CLOCK (dCLK), and CYCLE (CYC; homolog of mammalian BMAL1). dCLK and CYC are transcription factors of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) super- family that heterodimerize to stimulate the daily transcription of dper and tim, in addition to other clock and downstream genes. dPER plays a pivotal role in driving cyclical gene expression by undergoing daily translocation from the cytoplasm to the nucleus, where it functions as a critical nexus in the phase-specific inhibi- tion of dCLK-CYC transcriptional activity. Kinases are key players in controlling when in a daily cycle dPER engages in autoinhibi- tion by regulating its stability, timing of nuclear entry, and dura- tion in the nucleus, and possibly its repressor potency (reviewed in reference 3). Much progress has been made in understanding the role of phosphorylation in regulating dPER’s daily life cycle. At midday, dper and tim mRNA levels begin to rise, but dPER and TIM pro- tein levels remain low during the day. The instability of dPER is due mainly to phosphorylation by the DOUBLETIME (DBT; Dro- sophila homolog of CK1/ε) kinase (8, 9), whereas TIM is de- graded in a light-mediated pathway that involves the circadian photoreceptor CRYPTOCHROME (CRY) (reviewed in reference 10). After nightfall, TIM levels increase, and this enhances the interaction with dPER, which protects dPER against DBT-medi- ated degradation. In addition, the interaction of dPER and TIM promotes the translocation of both (in addition to PER-bound DBT) from the cytoplasm to the nucleus, an event that occurs Received 7 July 2015 Returned for modification 4 August 2015 Accepted 2 December 2015 Accepted manuscript posted online 28 December 2015 Citation Yildirim E, Chiu JC, Edery I. 2016. Identification of light-sensitive phosphorylation sites on PERIOD that regulate the pace of circadian rhythms in Drosophila. Mol Cell Biol 36:855– 870. doi:10.1128/MCB.00682-15. Address correspondence to Isaac Edery, edery@cabm.rutgers.edu. * Present address: Evrim Yildirim, Department of Neurobiology, Northwestern University, Evanston, Illinois, USA; Joanna C. Chiu, Department of Entomology and Nematology, University of California, Davis, California, USA. Copyright © 2016, American Society for Microbiology. 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