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

描述（由申请人提供）：家族性睡眠阶段提前综合征（FASPS）是人类昼夜节律系统中唯一已知的孟德尔表型。我们已经确定并描述了临床表型，并确定了五种基因，当突变时，导致FASPS。其中两个，酪蛋白激酶I？& ?（CKI?/?），被认为在家族中携带与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影响CKI?/？磷酸化的网站。在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，一种全面和公正的鉴定底物的方法是不可能的。我们将采用一种创新的化学遗传方法，通过工程突变进入ATP结合口袋来特异性标记这些酶的底物。ATP的相互化学修饰被设计成合成ATP类似物，这些类似物只能被突变的（类似物敏感的）激酶容纳。这将为CKI提供一个更完整的底物目录。并且将允许评估每个酶的冗余和独特功能。这种方法也将应用于鉴定这些激酶在已知底物上的多个磷酸化位点。体外生化分析可以用来监测FASPS突变对这些底物的具体影响。接下来，将产生转基因小鼠，使其携带含有类似物敏感突变的每个基因的BAC。这些将被交叉到空白背景上，并将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ATP中的磷酸基团。携带CKI和CKI类似物敏感突变的小鼠？长江基建?将生成。然后，我们可以通过使用化学抑制剂，在类似物敏感的ATP结合位点特异性结合，快速和可逆地灭活这些激酶。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，我们将关注昼夜节律系统，但也对CKI是否具有致命性感兴趣？敲除小鼠是其对发育的影响或其在小鼠整个生命周期中的活动的结果。这项工作将导致许多CKI的鉴定？这些激酶在人类昼夜节律中的作用的底物和分子解剖。鉴定底物和解剖生理节律等表型的特定途径将对生理节律表型的治疗和生理机制的理解产生深远的影响。

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.