Defining novel targets and functions of the protein kinase DYRK2
Protein post-translational modifications (PTMs) are an essential and conserved mechanism that allows eukaryotic cells to coordinate rapid responses to changing environmental conditions. Protein kinases, comprising a large eukaryotic superfamily of ~518 proteins, catalyse protein phosphorylation. Dual-specificity tyrosine-phosphorylation regulated kinases (DYRKs) are a family of protein kinases that are distantly related to the mitogen-activated protein kinases (MAPKs) and cyclin-dependent protein kinases (CDKs). Hundreds of substrates and interacting partner proteins have been identified for MAPKs and CDKs, whereas relatively little is known about the substrates and functions of DYRKs. DYRKs are evolutionarily conserved in protists, plants, fungi and animals; they widely function in cell cycle regulation, cytokinesis and cell differentiation, but they also orchestrate signalling responses to nitrosative, oxidative, nutritional, heat and osmotic stress. The five mammalian DYRKs can be divided into two subgroups based on their sequence homology: class I (DYRK1A, DYRK1B) and class II (DYRK2, DYRK3, DYRK4). Remarkable effort has been spent on identifying the roles of human DYRK1A, a Down syndrome candidate gene that is strongly implicated in neurogenesis, yet the physiological functions and substrates of the remaining human DYRK kinases are poorly characterised. This project employed a top-down experimental design with three main objectives: 1) assessing global cellular functions of DYRK2 using proteomic and transcriptomic analyses to identify protein and gene targets that are downstream of DYRK2; 2) validating novel DYRK2 substrates and localising the phospho-acceptor site(s) on target proteins using molecular biology and biochemical assays and 3) elucidating the biochemical purpose of DYRK2-substrate interactions identified in earlier experiments using DYRK2 knockout cells generated by CRISPR-Cas9 technology. Since DYRKs undergo cis-autoactivation during translation, a tetracycline-inducible expression system was employed to drive de novo transcription and translation of DYRK2 in HEK293 cells and to induce the serine/threonine kinase activity of DYRK2 against its target substrates. The DYRK2-inducible cells were used in combination with stable isotope labelling of amino acids in cell culture (SILAC), phosphopeptide enrichment and liquid chromatography tandem-mass spectrometry to identify phosphorylation events that were differentially upregulated following DYRK2 expression and represented candidate DYRK2 substrates. Follow-up work focused on the regulation of the autophagy cargo receptor p62 at distinct phosphorylation sites that may affect its oligomerisation capacity, as well as the regulation of the small ribonucleoprotein complex protein snRNP70 and possible effects on splice site recognition. The Hippo signalling pathway is a cascade that represses growth in response to environmental cues, such as increased cell-to-cell contacts, and here, novel links between the two classes of DYRKs and the Hippo signalling pathway were uncovered. A brief characterisation of DYRK2 knockout cells also showed that cells deficient in DYRK2 had increased mitotic missegregation events and DNA damage signalling, suggesting the presence of increased unrepaired or misrepaired double-strand breaks in DNA. These effects were thought to be independent of DYRK2 kinase activity since a predicted catalytically inactive DYRK2-expressing clone resembled wild-type DYRK2 cells. The work presented in this thesis provides novel insights into understudied areas of DYRK biology and highlights clear functional overlaps and differences between class I and class II DYRKs. Ultimately, determining normal physiological roles of DYRKs is key to understanding how their deregulation may contribute to a number of disease phenotypes, including cancer, neurodegenerative diseases and metabolic syndrome.
DYRK, DYRKs, DYRK2, Hippo signalling, Hippo, YAP, p62, SQSTM1, phosphorylation, substrates, RNA processing, CRISPR, Mass spectrometry, protein kinase, kinase, DYRK1B, DYRK1A, DYRK3, DYRK4, SILAC, Mitosis, DNA damage, Screening
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