Function of Casein Kinase 2 in the Circadian Clock

描述(申请人提供)：日常节律和行为部分由生物钟驱动。临床研究表明，昼夜节律缺陷会导致某些形式的抑郁症和睡眠障碍。对分子时钟机制的大部分了解都来自于对果蝇的遗传学研究。对昼夜节律基因的分析表明，转录反馈环是昼夜节律起搏器的核心。苍蝇的哺乳动物基因也与生物钟密切相关，使苍蝇成为发现生物钟基因的重要模式系统。蛋白质磷酸化越来越被认为对生物钟的计时很重要。有趣的是，人类苍蝇节律基因周期的同源突变会导致睡眠障碍，突变的蛋白质是磷酸化的缺陷底物。最近的工作已经确定了蛋白激酶酪蛋白激酶2(CK2)在果蝇生物钟中的一个新角色。CK2在体外可以直接磷酸化昼夜节律蛋白，周期蛋白(PER)和无时间蛋白(TIM)，突变体中PER和TIM蛋白的消失被推迟，提示该蛋白参与调节PER和TIM的稳定性。为了确定昼夜节律功能是否需要CK2α，将进行遗传筛选以确定CK2α的零等位基因。这样的筛选还可以识别CK2功能的修饰物。CK2α亚细胞定位的昼夜节律将被研究。为了探讨CK2α在PER和TIM磷酸化中的体内作用，将对纯合CK2α突变体的磷酸化状态和稳定性进行检测。将进行免疫共沉淀研究和寻找体外和体内的磷酸化位点，以建立直接的功能相互作用。令人惊讶的是，在起搏器神经元的轴突和终末中，CK2α也与关键的昼夜节律神经肽色素分散因子(PDF)共表达，这表明中央起搏器与神经元输出之间存在新的联系。CK2在调节PDF表达和酰胺化的各个方面的作用将被讨论。此外，还将使用电子显微镜检查CK2与PDF的关联。CK2在植物、动物和真菌中具有昼夜节律功能的发现表明，这些研究将与包括人类在内的许多生物系统相关。

DESCRIPTION (provided by applicant): Daily rhythms and behavior are driven in part by biological clocks. Clinical studies suggest that circadian rhythm defects contribute to certain forms of depression and sleep disorders. Much of the understanding of molecular clock mechanisms is derived from genetic studies in the fruit fly, Drosophila melanogaster. Analysis of circadian rhythm genes reveal that transcriptional feedback loops are at the core of circadian pacemakers. The mammalian counterparts of fly genes are also intimately involved in circadian clocks, establishing the fly as an important model system for clock gene discovery. Protein phosphorylation is increasingly being appreciated as important for the timing of biological clocks. Interestingly, a mutation in the human ortholog of the fly rhythm gene period leads to a sleep disorder in which the mutant protein is a defective substrate for phosphorylation. Recent work has identified a novel role for the protein kinase, casein kinase 2 (CK2), in the Drosophila clock. CK2 can directly phosphorylate the circadian rhythm proteins, PERIOD (PER) and TIMELESS (TIM) in vitro and PER and TIM protein disappearance is delayed in CK2 mutants, implicating this kinase in regulating PER and TIM stability. To determine if CK2 alpha is required for circadian function, genetic screens will be performed to identify null alleles of CK2 alpha. Such screens may also identify modifiers of CK2 function. The circadian regulation of CK2 alpha subcellular localization will be investigated. To address the in vivo role of CK2 alpha in PER and TIM phosphorylation, phosphorylation state as well as stability will be examined in homozygous CK2 alpha mutants. Coimmunoprecipitation studies and searches for in vitro and in vivo phosphorylation sites will be performed to establish direct functional interactions. Surprisingly, CK2 alpha is also co-expressed with the key circadian neuropeptide PIGMENT DISPERSING FACTOR (PDF) in the axons and termini of pacemaker neurons, suggesting a novel link between the central pacemaker and neuronal output. The role of CK2 in regulating various aspects of PDF expression and amidation will be addressed. In addition, the association of CK2 with PDF will be examined using electron microscopy. The discovery of a circadian function of CK2 in plants, animals, and fungi suggests these studies will be relevant to many biological systems, including humans.