The overall goal of this proposal is to better understand the multiple roles of protein phosphorylation in regulating circadian rhythms by focusing on PERIOD (PER), a core transcriptional repressor of the circadian transcriptome and the key biochemical timer that underlies animal circadian time-keeping mechanisms. In animal clocks, de novo synthesized PER goes through a dynamic multi-site phosphorylation program that is dependent on the activities of a number of kinases and phosphatases. Through evolutionary fine-tuning, completion of this phosphorylation cycle requires the duration of a circadian day, thereby closely linking PER phosphorylation program to the speed of the clock. The phase-specific phosphorylation program of PER provides sophisticated time-of-day specific modulations to its functional properties, including stability, subcellular localization, and transcriptional activity, thereby controlling the time and duration for which PER functions as a repressor of the circadian transcriptome. We have previously performed mechanistic studies on specific PER phosphorylation sites that are dependent on DOUBLETIME (DBT) and NEMO kinase activities, and found that they are integral part of a phosphorylation circuitry that sets te pace of the clock. In order to fully comprehend the role of DBT and NEMO-dependent PER phosphorylation in circadian biology, we are proposing to use Drosophila melanogaster as a model to address three main questions. (i) What are the sites that are modified by DBT and NEMO? (ii) What is the temporal progression of the phosphorylation program? (iii) What is the function of specific phosphorylation events? Our specific aims are to (1) map DBT- and NEMO-dependent PER phosphorylation sites using quantitative mass spectrometry;(2) decipher the temporal progression of the PER phosphorylation program in whole animals using phosphospecific antibodies in combination with laser scanning confocal microscopy;and (3) assay changes in PER interactome with respect to kinase activities and time using AP-MS (Affinity Purification and Mass Spectrometry) in order to generate new hypotheses regarding temporal changes in PER functional properties. By integrating the results from our three aims, we will understand how dynamic phase-specific phosphorylation of Drosophila PER progresses throughout the circadian day and how specific PER phosphorylation states impacts its role in circadian time-keeping. Results from our studies could pave the way for development of small molecule therapeutics and have profound impact on treatment of human disorders associated with phosphorylation defects, e.g. Familial Advanced Sleep Phase Syndrome (FASPS).
The overall goal of this proposal is to better understand the multiple roles of protein phosphorylation in regulating the function of (PERIOD) PER as a biochemical timer in animal circadian time-keeping. Since defects in PER protein phosphorylation has been linked to human health disorders, e.g. Familial Advanced Sleep Phase Syndromes (FASPS), our studies and new technologies we are developing could pave the way for high-throughput screening of small molecule pharmacological agents and development of drugs to correct sleeping disorders and other disorders with basis in circadian protein phosphorylation.
|Murphy, Katherine A; Tabuloc, Christine A; Cervantes, Kevin R et al. (2016) Ingestion of genetically modified yeast symbiont reduces fitness of an insect pest via RNA interference. Sci Rep 6:22587|
|Yildirim, Evrim; Chiu, Joanna C; Edery, Isaac (2016) Identification of Light-Sensitive Phosphorylation Sites on PERIOD That Regulate the Pace of Circadian Rhythms in Drosophila. Mol Cell Biol 36:855-70|
|Fu, Jingjing; Murphy, Katherine A; Zhou, Mian et al. (2016) Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD. Genes Dev 30:1761-75|
|Maliti, Deodatus Vincent; Marsden, C D; Main, B J et al. (2016) Investigating associations between biting time in the malaria vector Anopheles arabiensis Patton and single nucleotide polymorphisms in circadian clock genes: support for sub-structure among An. arabiensis in the Kilombero valley of Tanzania. Parasit Vectors 9:109|
|Kwok, Rosanna S; Lam, Vu H; Chiu, Joanna C (2015) Understanding the role of chromatin remodeling in the regulation of circadian transcription in Drosophila. Fly (Austin) 9:145-54|
|Kwok, Rosanna S; Li, Ying H; Lei, Anna J et al. (2015) The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila. PLoS Genet 11:e1005307|
|Svetec, Nicolas; Zhao, Li; Saelao, Perot et al. (2015) Evidence that natural selection maintains genetic variation for sleep in Drosophila melanogaster. BMC Evol Biol 15:41|