Circadian clocks regulate molecular oscillations that manifest into physiological and behavioral rhythms. Despite the prominent role played by rhythmic mRNA expression in circadian oscillators, time-of-day specific changes in clock protein levels are also critical for the normal progression of circadian pacemakers. Posttranslational mechanisms have emerged as significant contributors to the temporal regulation of clock protein levels and function. A conserved feature of animal clocks is that PERIOD (PER) proteins undergo daily oscillations in levels and phosphorylation states. In Drosophiia, PER protein phosphorylation states are controlled by kinases including DOUBLETIME (DBT) [casein kinase le ( C K I E in mammals)] and CK2, as well as protein phosphatase 1 (PP1) and 2A (PP2A). Hyperphosphorylated PER proteins are eventually targeted to the 26S proteasome by the F-box protein SLIMB (a homolog of p-TrCP in mammals). The timeof- day specific phosphorylation states of PER proteins not only regulate PER protein stability, but also affect subcellular localization, transcriptional inhibitor activity, and protein-protein interactions. The overall goal of this proposal is to better understand the contribution of clock protein phosphorylation and degradation pathways in regulating circadian rhythms by focusing on dPER, a central clock protein. By using Drosophiia melanogaster as a model, I propose to (1) examine the phosphorylation profile of PER proteins isolated under different physiologically relevant conditions by mass spectral analysis and characterize the function of dPER phosphorylation events using S2 cell culture and in vivo transgenic animal approaches. Mulitple dPER phosphorylation site clusters have been successfully identified using this approach. Of which, we identified the cluster centered around dPER(S47) as key phosphodeterminants that triggers PER-SLIMB binding. We are currently analyzing other clusters for their role in circadian timekeeping. Furthermore, I propose to (2) dissect the SLIMB-mediated dPER degradation pathway, especially focusing on the steps post-SLIMB binding. Finally, I plan to (3) characterize novel dPER-interacting proteins that was isolated using one-step or tandem purification, and determine their role in dPER phosphorylation, metabolism, and function.
; The Study of clock protein phosphorylation and metabolism has significant implications to public health. Mutations altering human PER2 phosphorylation or CKI kinase activity are linked to familial sleep disorders in humans. Taking a wider perspective, defects in circadian clock proteins have been implicated in other human disorders, including chronic sleep problems in the elderly, seasonal affective disorders, metabolic syndromes and susceptibility to drug and alcohol and cancer.
