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 (≅24 hr) clocks in animals are PERIOD (PER) proteins, transcriptional regulators that by means of complex multi-site phosphorylation programs undergo daily changes in levels and nuclear accumulation. Herein, 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 CK1δ/ϵ), the key circadian kinase regulating the daily changes in PER stability and phosphorylation. Mutant flies where phosphorylation at Ser826/Ser828 is blocked manifest behavioral rhythms with periods slightly longer then 1 hour and 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 phospho-sites on PER proteins can modulate the pace of downstream behavioral rhythms without altering central aspects of the clock mechanism.