Cycling of Circadium Rhythm Proteins

描述(由申请者提供)：长期目标是了解昼夜节律(大约24小时)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，在睡眠-觉醒周期、体温、激素分泌、血压和肝脏新陈代谢中可以看出昼夜周期。生物钟功能紊乱与睡眠障碍以及肿瘤生长等其他病理因素有关。内源性生物钟的分子性质在很大程度上是通过对果蝇黑腹果蝇的研究确定的。这些研究表明，时钟是由一个更多的反馈环组成的，在这些反馈环中，特定基因(即所谓的时钟基因)的蛋白质产物以昼夜节律的方式调节自己的mRNA的合成。结果，这些mRNAs和蛋白质的循环表达，从而维持了下游生理成分的表达。我们发现，即使编码果蝇时钟蛋白的mRNAs不循环，果蝇时钟蛋白周期(PER)和无时间(TIM)的周期性表达也会发生，并且节律性蛋白表达足以驱动节律性行为。在哺乳动物中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的激酶似乎不会循环。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节亚基wdb和tws以昼夜节律的方式循环。对这些亚基或PP2A催化亚基的操纵会导致分子时钟和行为节律的缺陷。此外，PP2A在体外可直接使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亚单位在苍蝇头部表达的时间和地点，并确定它们在一天中不同时间的亚细胞分布，因为Per-Tim的核定位显示出昼夜节律。(2)确定哪个PP2A亚基介导了磷酸酶复合体对PER的不同影响。(3)获得和鉴定wdb和tws的附加突变体。(4)确定时钟如何控制TWS的循环，并解决此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.