Function of Casein Kinase 2 in the Circadian Clock

酪蛋白激酶 2 在生物钟中的功能

• 批准号: 6711748
• 负责人: Ravi Allada
• 金额: $ 35.05万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6711748
• 关键词: Drosophilidae; arthropod genetics; biological clocks; casein kinase; chemical stability; circadian rhythms; electron microscopy; enzyme mechanism; genetic screening; mutant; neuropeptides; phosphorylation

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.

描述（由申请人提供）：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。对分子钟机制的理解大多来自果蝇的遗传研究。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。果蝇基因的哺乳动物对应物也密切参与生物钟，建立了果蝇作为时钟基因发现的重要模型系统。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，果蝇节律基因周期的人类直系同源物中的突变导致睡眠障碍，其中突变蛋白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），在果蝇时钟的一个新的作用。CK2在体外可直接磷酸化昼夜节律蛋白PERIOD（PER）和TIMELESS（TIM），并且在CK2突变体中PER和TIM蛋白的消失被延迟，暗示该激酶在调节PER和TIM稳定性中起作用。为了确定昼夜节律功能是否需要CK2 α，将进行遗传筛查以鉴定CK2 α的无效等位基因。这样的筛选也可以鉴定CK2功能的修饰剂。将研究CK2 α亚细胞定位的昼夜节律调节。为了阐明CK2 α在PER和TIM磷酸化中的体内作用，将在纯合CK2 α突变体中检查磷酸化状态以及稳定性。将进行免疫共沉淀研究和体外和体内磷酸化位点的搜索，以建立直接的功能相互作用。令人惊讶的是，CK2 α也与关键的昼夜神经肽色素分散因子（PDF）在起搏神经元的轴突和末端共表达，这表明中枢起搏器和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得到解决。此外，将使用电子显微镜检查CK2与PDF的关联。CK2在植物、动物和真菌中的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。