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: 7687486
• 负责人: YING-HUI FU
• 金额: $ 31.67万
• 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/7687486
• 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）：家族性高级睡眠阶段综合症（FASPS）是人类昼夜节律系统唯一已知的孟德尔表型。我们已经鉴定和表征了临床表型，并鉴定了五个突变时会导致 FASPS 的基因。其中两个，酪蛋白激酶 I？ &? (CKI?/?)，被认为含有与 FASPS 在家族中分离并导致体外活性降低的突变。第三个基因（第 2 期）的突变会影响 CKI？/？磷酸化位点。许多实验室的工作已经表征了这些激酶的一些底物，但鉴于任何细胞或生物体中存在大量激酶和磷酸酶，不可能采用全面且公正的方法来识别底物。我们将采用创新的化学遗传方法，通过将突变引入 ATP 结合袋来特异性标记这些酶的底物。 ATP 的相互化学修饰被设计为合成只能由突变（类似物敏感）激酶容纳的 ATP 类似物。这将为 CKI?/? 提供更完整的底物概要。并将允许评估每种酶的冗余和独特功能。该方法还将应用于识别这些激酶已知底物上的多个磷酸化位点。可以进行体外生化测定来监测 FAPSS 突变对每种底物的具体影响。接下来，将产生携带 BAC 的转基因小鼠，每个基因都含有类似物敏感突变。这些将与空背景交叉，并代表具有接近正常激酶活性的小鼠，因为类似物敏感激酶仍然接受并转移来自 ATP 的磷酸基团。携带两种 CKI 类似物敏感突变的小鼠？和长江基建？将被生成。然后，我们可以通过使用特异性结合类似物敏感 ATP 结合位点的化学抑制剂来快速、可逆地灭活这些激酶。将在不同的发育时间点对这些小鼠进行研究，以监测一种或两种激酶失活时的表型。我们特别关注昼夜节律系统，但也对 CKI 中所见的致命性是否感兴趣？基因敲除小鼠是其对小鼠发育或其整个生命周期活动的影响的结果。这项工作将导致许多 CKI?/? 的识别。这些激酶在人类昼夜节律中的作用的底物和分子解剖。识别底物并剖析表型（例如昼夜节律）中的特定途径将对昼夜节律表型的治疗和生理机制的理解产生深远的影响。 公共卫生相关性：CKI？和长江基建？是许多重要生物学功能的重要激酶。该提案概述了阐明CKI正常作用的计划？和长江基建？通过识别其底物和旨在了解对 CKI?/? 功能后果重要的底物的研究在昼夜节律中。我们还将检查体内可逆地灭活这些激酶所产生的表型。