The long-term goals are to understand the molecular basis of circadian (~24 hour) rhythms. These rhythms are controlled by clocks endogenous to most organisms and are manifest in many different physiological processes. Disrupted functioning of clocks has been associated with sleep disorders as well as other pathologies such as tumor growth. The molecular nature of the endogenous circadian clock was determined largely through studies done in the fruit fly, Drosophila melanogaster. These studies showed that the clock is composed of specific genes (so-called """"""""clock genes"""""""") whose protein products regulate the synthesis of their own mRNAs at a specific time of day. The feedback loop thus generated drives the cycling of downstream physiological components which, in turn, drive rhythms of behavior and physiology. However, the mechanisms that maintain such feedback loops with a 24 hour periodicity are not understood. In addition, rhythms are often maintained under conditions where levels of some clock mRNAs, and sometimes even clock proteins, are held constant, indicating the critical role of post-translational regulation. Synchrony of the Drosophila clock to light also relies upon such post-translational mechanisms. We hypothesize that it is the feedback activity of clock proteins that must cycle in order to maintain clock function, and that the cycling of this activity is driven by temporal control of protein stability, nuclear expression and ability to repress transcription. These features of clock proteins are affected, to a large extent, by phosphorylation, but key regulatory steps have not been identified. We propose to use tools we have recently discovered/generated to dissect the post-translational regulation of the major cycling Drosophila proteins, period (PER) and timeless (TIM). We will also investigate the clock's response to light, which has its basis in the control of protein stability.
Specific aims are to: (1) Determine the mechanisms underlying the behavioral phenotype of a novel tim allele We have identified a novel mutation of tim which affects the stability and nuclear localization of PER and TIM and the phosphorylation of PER. This provides us with a powerful tool to address the mechanisms underlying nuclear expression, and to determine the effect of subcellular localization on phosphorylation and stability;(2) Identify the functional significance of novel phosphorylation sites on PER and TIM. We have identified novel phosphorylation sites on PER and TIM through mass spectrometry analysis. We will investigate the role of these sites in the processes listed above;(3) Identify kinases required for the TIM response to light. Through a small molecule inhibitor screen, we have identified classes of kinase required for TIM degradation by light. We will identify the specific kinases involved.

Public Health Relevance

Circadian rhythms pervade all aspects of physiology and behavior, such as sleep-wake, body temperature, hormonal secretions, blood pressure and liver metabolism. Disruption of these rhythms, which may be caused by many conditions including shift work, has been associated with metabolic disorders, sleep problems and even tumor growth (Boivin et al., 2007;Fu et al., 2002;Toh et al., 2001;Xu et al., 2005). An understanding of the molecular mechanisms that drive these rhythms will lead to the development of treatments for rhythm dysfunction.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Biological Rhythms and Sleep Study Section (BRS)
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Mitler, Merrill
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University of Pennsylvania
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