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

描述(由申请人提供)：家族性晚期睡眠相综合征(FASPS)是人类昼夜节律系统中唯一已知的孟德尔表型。我们已经确定并表征了临床表型，并确定了五个基因，当突变时，会导致FASPS。其中两个，酪蛋白激酶I？&？(CKI？/？)，被认为在家族中含有与FASPS分离的突变，并导致体外活性降低。第三个基因的突变，周期2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，但由于任何细胞或生物体中都存在大量的激酶和磷酸酶，因此不可能有一种全面和公正的方法来识别底物。我们将采用一种创新的化学遗传方法，通过将突变工程到ATP结合口袋中，专门标记这些酶的底物。ATP的相互化学修饰被设计成合成只能被突变的(类似物敏感的)激酶调节的ATP类似物。这将为长江基建提供更完整的底物概要。并将允许评估每种酶的多余和独特的功能。这一方法也将被应用于通过这些激酶识别已知底物上的多个磷酸化位点。可以进行体外生化分析来监测FASPS突变对每种底物的特定影响。接下来，将产生携带BAC的转基因小鼠，每个基因都含有模拟敏感突变。这些将被交叉到零背景上，并将代表具有接近正常的激酶活性的小鼠，因为类比敏感的激酶仍然接受并转移来自ATP的磷酸基团。携带CKI？和CKI？长江基建呢？将会被生成。然后，我们可以通过使用化学抑制剂来快速和可逆地灭活这些激酶，这些抑制剂专门结合在类似物敏感的ATP结合部位。这些小鼠将在不同的发育时间点进行研究，以监测其中一个或两个激酶失活时的表型。特别是，我们将专注于昼夜节律系统，但也对长江基建中看到的致命性是否感兴趣？敲除老鼠是由于其对发育的影响或其在小鼠整个生命周期中的活动所致。这项工作将导致识别许多CKI？/？这些激酶在人体昼夜节律性中作用的底物和分子剖析。识别表型的底物并剖析特定的途径，如昼夜节律性，将对昼夜表型的治疗和生理机制的理解具有深远的意义。 公共卫生相关性：长江基建？长江基建呢？是许多基本生物学功能的重要激酶。这项建议概述了一个计划，以阐明长江基建的正常角色？长江基建呢？通过鉴定它们的底物和旨在了解对CKI功能结果重要的底物的研究。以昼夜节律。我们还将研究体内可逆性失活这些激酶的表型。

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.