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 PERIOD (PER), the central clock protein. In animal clocks, PER undergoes daily oscillations in levels and phosphorylation states that are regulated by DOUBLETIME (DBT in Drosophila, CKIe in mammals), CKII, and phosphatase 2A. Hyperphosphorylated PER proteins are eventually targeted to the 26S proteasome by the F-box protein SLIMB (a homolog of B-TrCP in mammals). My immediate goal is to complete an additional 2 years of mentored research in Dr. Isaac Edery's lab. My long term goal is to become a tenure-tracked independent scientist at an academic institution, and contribute to a better understanding of how circadian oscillations are generated and maintained, and how they control the timing of output physiological processes. During the mentored phase, I plan to examine the mechanisms by which different phosphorylation events, identified from a mass spectral analysis of DBT-phosphorylated PER proteins, regulate PER function, subcellular localization, and interaction with other clock proteins. In addition, to better understand the SLIMB-mediated PER degradation pathway, and to build on my studies on PER-SLIMB interaction, I plan to dissect the steps of SLIMB-mediated PER degradation pathway following PER-SLIMB interaction. Upon transition into the independence phase, I propose to examine the phosphorylation profile of PER proteins isolated under other physiologically relevant conditions by mass spectral analysis, and examine the functional significance of the identified phosphosites in vivo. I believe this systematic approach will yield significant insights into the regulation of PER function by phosphorylation. Finally, I propose to use tandem affinity purification to isolate novel clock components and protein interactions that regulate PER phosphorylation, metabolism, and function. Relevance: 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 addiction, and cancer.
