Cycling of Circadian Rhythm Proteins

描述（由申请人提供）：长期目标是了解昼夜（约24小时）节律的分子基础。这些节律由大多数生物体内源性的时钟控制，并在许多不同的生理过程中表现出来。例如，在人类中，昼夜节律的周期性在睡眠-觉醒周期、体温、激素分泌、血压和肝脏代谢中是可辨别的。生物钟功能的中断与睡眠障碍以及其他病理学（如肿瘤生长）有关。内源性生物钟的分子本质主要是通过对果蝇（Drosophila melanogaster）的研究确定的。这些研究表明，生物钟是由一个或多个反馈回路组成的，其中特定基因（所谓的“时钟基因”）的蛋白质产物以昼夜节律的方式调节其自身mRNA的合成。因此，维持了这些mRNA和蛋白质的循环表达，从而维持了下游生理组分的循环表达。我们发现，周期表达的果蝇时钟蛋白周期（PER）和无时间（TIM），即使发生时，编码它们的mRNA不循环和有节奏的蛋白质表达是足以驱动节奏的行为。对哺乳动物的研究证实了时钟蛋白循环的相对RNA独立性，这很可能是通过导致周期性周转的节律性磷酸化事件实现的。然而，磷酸化PER和TIM的激酶似乎不循环。我们最近发现PP 2A磷酸酶家族影响PER的稳定性和核表达，并且该家族的两个调节亚基WDB和TWS以昼夜节律循环。操纵这些亚基或PP 2A的催化亚基会导致分子钟和行为节律的缺陷。此外，PP 2A在体外直接使PER脱磷酸化。我们建议确定PP 2A在生物钟中的作用。我们将（1）确定PP 2A亚基在苍蝇头中表达的时间和位置，并确定它们在一天中不同时间的亚细胞分布，因为PER-TIM的核定位显示昼夜节律。(2)确定哪个PP 2A亚基介导磷酸酶复合物对PER的不同作用。(3)产生和表征wdb和tws的额外突变体。(4)确定生物钟如何控制tws的循环，并说明这种循环与行为节律的相关性。

DESCRIPTION (provided by applicant): The long-term goals are to understand the molecular basis of circadian (approximately 24 hour) rhythms. These rhythms are controlled by clocks endogenous to most organisms and are manifest in many different physiological processes. In humans, for instance, a circadian periodicity is discernible in sleep-wake cycles, body temperature, hormonal secretions, blood pressure and liver metabolism. 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 one of more feedback loops in which protein products of specific genes (so-called "clock genes") regulate the synthesis of their own mRNAs in a circadian fashion. As a result, cyclic expression of these mRNAs and proteins, and thereby of downstream physiological components, is maintained. We found that cyclic expression of the Drosophila clock proteins period (PER) and timeless (TIM), occur even when the mRNAs encoding them do not cycle and that rhythmic protein expression is sufficient to drive rhythmic behavior. Studies in mammals corroborate the relative RNA-independence of clock protein cycling which is most likely achieved through rhythmic phosphorylation events that lead to periodic turnover. However, the kinases that phosphorylate PER and TIM do not appear to cycle. We have recently found that the PP2A phosphatase family affects stability and nuclear expression of PER and that two of the regulatory subunits of this family, wdb and tws, cycle with a circadian rhythm. Manipulations of either of these subunits or of the catalytic subunit of PP2A results in defects in the molecular clock and in behavioral rhythms. In addition, PP2A directly dephosphorylates PER in vitro. We propose to determine the role of PP2A in the circadian clock. We will (1) Determine when and where PP2A subunits are expressed in the fly head and also determine their subcellular distribution at different times of day, given that the nuclear localization of PER-TIM displays a circadian rhythm. (2) Determine which PP2A subunit mediates each of the different effects of the phosphatase complex on PER. (3) Generate and characterize additional mutants of wdb and tws. (4) Determine how the clock controls the cycling of tws and address the relevance of this cycling for behavioral rhythms.