A chemical genetic approach to dissect CKId & CKIe function in circadian rhythm

剖析 CKId 的化学遗传学方法

• 批准号: 8121600
• 负责人: YING-HUI FU
• 金额: $ 31.04万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8121600
• 关键词: Advanced Sleep Phase Syndrome; Affect; Alleles; Asthma; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; Birds; Cells; Chemicals; Circadian Rhythms; Classic Migraine; Complement; Development; Dissection; Engineering; Enzymes; Family; Functional disorder; Generations; Genes; Human; In Vitro; Individual; Investigation; Knock-out; Knockout Mice; Label; Laboratories; Lead; Life; Lysine; Methods; Modeling; Modification; Molecular; Monitor; Mus; Mutate; Mutation; Newborn Infant; Organism; Pathology; Pathway interactions; Pattern; Periodicity; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation Site; Phosphotransferases; Physiological; Regulation; Research Personnel; Role; Signal Transduction; System; Tetanus Helper Peptide; Therapeutic; Time; Transgenes; Transgenic Mice; Transgenic Organisms; Work; analog; casein kinase I; chemical genetics; clinical phenotype; design; enzyme substrate; falls; in vitro activity; in vivo; inhibitor/antagonist; innovation; inorganic phosphate; interest; mouse model; public health relevance; tool

DESCRIPTION (provided by applicant): Familial Advanced Sleep Phase Syndrome (FASPS) is the only known Mendelian phenotype of the human circadian system. We've identified and characterized the clinical phenotype and identified five genes that, when mutated, cause FASPS. Two of these, casein kinase I? & ? (CKI?/?), are recognized to harbor mutations that segregate with FASPS in families and lead to decreased activity in vitro. A mutation in a third gene, period 2 affects a CKI?/? phosphorylation site. Work in a number of laboratories has characterized some substrates of these kinases, but a comprehensive and unbiased method for identifying substrates has been impossible given the large number of kinases and phosphatases present in any cell or organism. We will employ an innovative chemical genetic approach to specifically label substrates of these enzymes by engineering mutations into the ATP binding pocket. Reciprocal chemical modifications of ATP are engineered to synthesize ATP analogs that can only be accommodated by the mutated (analog-sensitive) kinases. This will provide a more complete compendium of substrates for CKI?/? and will allow assessment of the redundant and unique functions of each enzyme. This approach will also be applied in identifying multiple phosphorylation sites on known substrates by these kinases. In vitro biochemical assays can be performed to monitor specific effects of the FASPS mutations on each of these substrates. Next, transgenic mice will be generated to carry a BAC with each gene harboring the analog-sensitive mutations. These will be crossed onto null backgrounds and will represent mice with near normal kinase activity since the analog-sensitive kinases still accept, and transfer phosphate groups from ATP. Mice carrying analog sensitive mutations for both CKI? and CKI? will be generated. We can then rapidly and reversibly inactivate these kinases through use of chemical inhibitors that bind specifically in the analog-sensitive ATP binding site. These mice will be studied at different developmental time points to monitor the phenotype when one or both kinases are inactivated. In particular, we will focus on the circadian system but are also interested in whether the lethality that is seen in the CKI? knock out mice is the result of its effects on development or of its activity throughout the life of the mouse. This work will result in identification of many CKI?/? substrates and molecular dissection of the role of these kinases in human circadian rhythmicity. Identification of substrates and dissection of particular pathways in phenotypes such as circadian rhythmicity will have profound implications for therapeutics of circadian phenotypes and understanding of physiological mechanisms. PUBLIC HEALTH RELEVANCE: CKI? and CKI? are important kinases for many essential biological functions. This proposal outlines a plan to elucidate the normal role of CKI? and CKI? through identification of their substrates and studies aimed at understanding substrates that are important for the functional consequences of CKI?/? in circadian rhythm. We will also examine phenotypes resulting from reversibly inactivating these kinases in vivo.

描述（由申请人提供）：家族性晚期睡眠期综合征（FASP）是人类昼夜节律系统的唯一已知的孟德尔表型。我们已经确定并表征了临床表型，并确定了五个引起FASP的基因。其中两个，酪蛋白激酶I？ ＆？ （CKI？/？），被公认为藏有家族中Fasps隔离的突变，并导致体外活性降低。第三基因中的突变，周期2会影响CKI？/？磷酸化位点。许多实验室的工作表征了这些激酶的某些底物，但是鉴于任何细胞或生物体中存在大量的激酶和磷酸酶，不可能是鉴定底物的全面且无偏见的方法。我们将采用一种创新的化学遗传方法，通过在ATP结合口袋中进行工程突变，以特异性标记这些酶的底物。 ATP的相互化学修饰是设计为合成ATP类似物的，这些ATP类似物只能由突变（模拟敏感）激酶适应。这将为CKI的基材提供更完整的汇编？/？并将允许评估每个酶的冗余功能。这种方法还将应用于这些激酶对已知底物的多个磷酸化位点。可以进行体外生化测定，以监测FASPS突变对每个底物的特定影响。接下来，将生成转基因小鼠，以携带每个基因携带模拟敏感的突变的BAC。这些将越过无效的背景，并将代表具有接近正常激酶活性的小鼠，因为模拟敏感激酶仍然接受，并从ATP转移磷酸基团。携带两个CKI的模拟敏感突变的小鼠？和CKI？将生成。然后，我们可以通过使用专门在模拟敏感的ATP结合位点上结合的化学抑制剂来快速和可逆地使这些激酶迅速失活。当一种或两种激酶失活时，将在不同的发育时间点进行研究，以监测表型。特别是，我们将重点关注昼夜节律系统，但也对CKI中看到的致命性感兴趣？敲除小鼠是其在小鼠整个生命中对发育或活性的影响的结果。这项工作将导致识别许多CKI？/？这些激酶在人昼夜节律中的作用的底物和分子解剖。在昼夜节律等表型中鉴定底物和特定途径的解剖将对昼夜节律表型的治疗和对生理机制的理解产生深远的影响。 公共卫生相关性：CKI？和CKI？是许多基本生物学功能的重要激酶。该提议概述了阐明CKI正常作用的计划？和CKI？通过鉴定其底物和研究，旨在理解对CKI的功能后果很重要的底物？/？以昼夜节律。我们还将检查由于体内使这些激酶不可逆地失活而导致的表型。