Cycling of Circadian Rhythm Proteins

描述（由申请人提供）：长期目标是了解昼夜节律（大约24小时）节奏的分子基础。这些节律是由大多数生物体的内源性时钟控制的，并在许多不同的生理过程中表现出来。例如，在人类中，睡眠-觉醒周期、体温、激素分泌、血压和肝脏代谢都有明显的昼夜周期。生物钟功能紊乱与睡眠障碍以及其他病理（如肿瘤生长）有关。内源性生物钟的分子性质主要是通过对果蝇（Drosophila melanogaster）的研究确定的。这些研究表明，生物钟是由多个反馈回路中的一个组成的，在这些反馈回路中，特定基因的蛋白质产物（所谓的“生物钟基因”）以昼夜节律的方式调节其自身mrna的合成。因此，这些mrna和蛋白质的循环表达，以及下游生理成分的循环表达得以维持。我们发现果蝇时钟蛋白周期（PER）和永恒（TIM）的循环表达即使在编码它们的mrna不循环的情况下也会发生，并且有节奏的蛋白质表达足以驱动有节奏的行为。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有节奏的磷酸化事件实现的。然而，磷酸化PER和TIM的激酶似乎没有循环。我们最近发现PP2A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控亚基wdb和tws具有昼夜节律循环。操纵这些亚基或PP2A的催化亚基都会导致分子钟和行为节律的缺陷。此外，PP2A在体外直接使PER去磷酸化。我们建议确定PP2A在生物钟中的作用。我们将(1)确定PP2A亚基在果蝇头部中的表达时间和位置，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)确定哪些PP2A亚基介导磷酸酶复合物对PER的不同作用。(3)生成wdb和tws的其他突变体并对其进行表征。(4)确定生物钟如何控制tws的循环，并解决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.