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

描述（由申请人提供）：家族性睡眠时相提前综合征（FASPS）是人类昼夜节律系统中唯一已知的孟德尔表型。我们已经鉴定并描述了临床表型，并确定了五个基因，当突变时，导致FASPS。其中两个酪蛋白激酶I？& ? (CKI?/?),在家族中与FASPS分离并导致体外活性降低的突变。第三个基因的突变，周期2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，但是考虑到任何细胞或生物体中存在大量的激酶和磷酸酶，用于鉴定底物的全面和无偏见的方法是不可能的。我们将采用一种创新的化学遗传方法，通过将突变工程化到ATP结合口袋中来特异性标记这些酶的底物。ATP的相互化学修饰被工程化以合成只能由突变的（类似物敏感的）激酶适应的ATP类似物。这将为CKI？/？提供更完整的底物纲要。并且将允许评估每种酶的冗余和独特功能。这种方法也将被应用于确定多个磷酸化位点上已知的底物由这些激酶。可以进行体外生物化学测定以监测FASPS突变对这些底物中的每一种的特定影响。接下来，将产生转基因小鼠，以携带具有携带类似物敏感突变的每个基因的BAC。这些将在空背景上交叉，并将代表具有接近正常激酶活性的小鼠，因为类似物敏感性激酶仍然接受并转移ATP的磷酸基团。携带CKI？而CKI？将被生成。然后，我们可以通过使用化学抑制剂快速可逆地抑制这些激酶，这些化学抑制剂特异性地结合在类似物敏感的ATP结合位点。这些小鼠将在不同的发育时间点进行研究，以监测一种或两种激酶失活时的表型。特别是，我们将重点放在昼夜节律系统，但也有兴趣在是否是在CKI看到的杀伤力？基因敲除小鼠是其对发育的影响或其在小鼠一生中的活性的结果。这项工作将导致许多CKI识别？/？底物和分子解剖的作用，这些激酶在人类昼夜节律。识别底物和解剖表型中的特定通路，如昼夜节律性，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：长江基建？而CKI？是许多基本生物功能的重要激酶。这份建议书概述了一项计划，以阐明长江基建的正常作用？而CKI？通过识别其底物和研究，旨在了解底物是重要的功能后果CKI？/？昼夜节律。我们还将研究可逆失活这些激酶在体内产生的表型。

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.