By means of endogenous circadian (approx. 24 hr) """"""""clocks"""""""" or pacemakers that can be synchronized to the daily and seasonal changes in external time cues (zeitgebers), most notably visible light and ambient temperature, life forms anticipate environmental transitions, perform activities at biologically advantageous times during the day and undergo characteristic seasonal responses. Tightly controlled oscillations in the levels of key clock proteins are essential for the normal progression of circadian clocks. Core features of many, if not all, circadian clocks are transcriptional feedback loops that generate daily cycles in the levels of clock mRNAs. However, posttranslational mechanisms make significant contributions to the temporal regulation of clock protein levels. Time-of-day specific differences in phosphorylation that result in phase-specific changes in protein stability appear to be a common strategy in generating daily cycles in clock protein abundance. The main theme of this proposal is to better understand the contribution of clock protein phosphorylation and its intersection with degradation pathways in regulating circadian rhythms. In particular, a conserved feature of animal clocks is that period (PER) proteins undergo daily rhythms in phosphorylation and levels that are regulated by casein kinase I epsilon (ckI epsilon). A major foundation for the proposed specific aims is based on our recent work showing that in D. melanogaster, phosphorylated PER is targeted to the 26S proteasome by the F-box protein Slimb. A multidimensional experimental plan that integrates in vitro strategies, tissue culture-based systems and whole animal approaches will be used to understand the role of phosphorylation in regulating PER metabolism, subcellular localization and function. How Slimb recognizes phosphorylated PER will be determined. This is an especially intriguing problem because PER does not contain a recognized Slimb binding region. Does multisite phosphorylation of suboptimal sites generate a phosphorylation threshold for binding? Other kinases and components of the ubiquitin/proteasome pathway that regulate PER phosphorylation and/or stability will be identified. A long-term goal is to apply similar experimental strategies to understand how dynamic changes in the levels/activities of all the key clock proteins in Drosophila are regulated and integrated to generate a self-sustaining oscillator. Abnormal PER phosphorylation is associated with variant human sleep behavior. In addition, PER proteins have a role in cancer and apoptosis. Thus, it is likely that the proposed studies will have broad significance for the understanding of PER function and clock mechanisms in humans. Also, it is anticipated that the proposed studies will provide novel insights into the rules of engagement underlying substrate recognition by F-box proteins. This could be particularly rewarding in the case of Slimb and its mammalian homolog beta-TrCP, which recognize a variety of phosphotargets and have important roles in development, apoptosis, inflammatory responses and cancer.
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