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

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.