Identification of Citron kinase substrates by using chemical genetics and quantitative phospho-proteomics

柠檬酸激酶(Citron Kinase，CIT-K)是一种多功能的丝氨酸/苏氨酸激酶，在细胞周期的不同方面发挥着重要作用，包括DNA损伤控制、有丝分裂纺锤体的定位和胞质分裂。CIT-K的突变与人类原发性小头畸形的发生有关，该激酶已被确定为癌症治疗的潜在靶点。尽管有证据表明，CIT-K的激酶结构域在细胞分裂过程中是胞质分裂和纺锤体定向所必需的，并且CIT-K的激酶活性的突变会导致人类原发性小头畸形，但到目前为止，只有一种骨化的CIT-K底物被鉴定出来。因此，需要一种更系统、更全面的方法来识别整个细胞周期中的CIT-K靶标，以了解CIT-K介导的磷酸化调节细胞周期并影响人脑发育的分子机制。为了解决这个问题，我们建议使用互补和收敛的实验策略，使用基因编辑、化学遗传学、定量磷酸蛋白质组学和免疫沉淀与质谱学(MS)相结合的方法。我们计划使用CRISPR/Cas9基因编辑来产生一个永生化的、未转化的RPE-1细胞系，该细胞系含有CIT-K‘类似物敏感’(AS)突变体。因为突变体可以接受‘大体积’的ATP类似物，并且可以被这些体积大的ATP类似物的非水解性形式选择性地抑制。这个CIT-KAS细胞系随后将被用两种平行和互补的方法来分析CIT-K的激酶活性的作用。通过使用基于SILAC的定量磷酸蛋白质组学，我们将在细胞周期的不同阶段同步存在或不存在大量ATP抑制剂的情况下，表征和比较该CIT-KAS细胞系的磷酸蛋白质组。这种方法将为我们提供响应CIT-K激活而被磷酸化的蛋白质的全球视角，但它最终不会告诉我们这些蛋白质是否是直接的CIT-K底物。因此，同时，我们还将使用该细胞系来硫代磷酸化CIT-K底物，方法是将来自相同细胞周期阶段的提取物与笨重的N6取代形式的ATP S孵育，然后使用硫代磷酸酯特异性抗体进行免疫沉淀，并通过MS鉴定。通过比较表达CIT-KAS或野生型CIT-K的细胞提取物的免疫沉淀物，将确定特定的底物。使用这两种方法确定的最有趣的CIT-K底物将在一系列实验中进行测试，以确认它们与CIT-K共定位，并可在体外被该激酶磷酸化。我的实验室过去已经使用过的技术，例如定向突变和产生磷酸化特异性抗体，然后将被用来理解这些CIT-K介导的磷酸化事件的作用。最后，我们将通过用体积庞大的抑制剂处理CIT-KAS细胞来研究CIT-K的激酶活性在有丝分裂中的作用，然后用时间推移和免疫荧光显微镜分析有丝分裂事件。

Citron kinase (CIT-K) is a multifunctional serine/threonine kinase that plays important roles in different aspects of the cell cycle, including DNA damage control, the orientation of the mitotic spindle and cytokinesis. Mutations in CIT-K have been linked to the development of human primary microcephaly and this kinase has been identified as a potential target in cancer therapy. Although compelling evidence indicates that the kinase domain of CIT-K is necessary for cytokinesis and spindle orientation during cell division and mutations impairing CIT-K's kinase activity cause human primary microcephaly, only one bone fide CIT-K substrate has been identified so far. Therefore, a more systematic and comprehensive approach is required to identify CIT-K targets throughout the cell cycle in order to understand the molecular mechanisms by which CIT-K-mediated phosphorylation regulates the cell cycle and affects human brain development. To address this, we propose to use complementary and convergent experimental strategies that employ a combination of gene editing, chemical genetics, quantitative phospho-proteomics and immuno-precipitation coupled with mass spectrometry (MS).We plan to use CRISPR/Cas9 gene editing to generate an immortalised, non-transformed RPE-1 cell line harbouring a CIT-K 'analogue sensitive' (AS) mutant. AS mutants can accept 'bulky' ATP analogues and can be selectively inhibited by non-hydrolysable forms of these bulky ATP analogues. This CIT-KAS cell line will then be utilised to dissect the role of CIT-K's kinase activity using two parallel and complementary approaches. By using SILAC-based quantitative phospho-proteomics, we will characterise and compare the phospho-proteomes of this CIT-KAS cell line in the presence or absence of bulky ATP inhibitors in cells synchronised at different stages of the cell cycle. This approach will provide us with a global view of the proteins that are phosphorylated in response to CIT-K activation, but it will not ultimately tell us if these proteins are direct CIT-K substrates. Thus, in parallel we will also use this cell line to thio-phosphorylate CIT-K substrates by incubating extracts from the same cell cycle stages with bulky N6-substituted forms of ATP S. Thio-phosphorylated proteins will then be immuno-precipitated using a thiophosphate ester-specific antibody and identified by MS. Comparison of immuno-precipitates from the pull downs of extracts from cells expressing either CIT-KAS or wild type CIT-K will identify specific substrates. The most interesting CIT-K substrates identified using these two methodologies will then be tested in a series of experiments to confirm that they co-localise with CIT-K and can be phosphorylated by this kinase in vitro. Techniques already used by my lab in the past, such as targeted mutagenesis and generation of phospho-specific antibodies, will then be employed to understand the role of these CIT-K-mediated phosphorylation events. Finally, we will investigate the role of CIT-K's kinase activity in mitosis by treating CIT-KAS cells with a bulky inhibitor and then analyse mitotic events by time-lapse and immuno-fluorescence microscopy.