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We have previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs), responsible for controlling numerous components critical to differentiated SMC function (Nakagaki-Silva et al., 2019). Therefore, the precise control of this splicing regulator is vital in determining SMC fate. Although RBPMS is transcriptionally downregulated during SMC dedifferentiation, protein activity is also expected to be directly regulated. Post-translational modifications, specifically phosphorylation, provide the cell with a highly dynamic and reversible mechanism to regulate the activity of proteins. Examining publicly available phosphoproteomic databases, I found several RBPMS residues reported to be phosphorylated. In particular, Thr113 and Thr118 are immediately adjacent to the RRM domain, and their phosphorylation could potentially affect RNA-binding, nuclear localization, dimerization, higher-order oligomerization, and splicing regulatory activity. I, therefore, created a series of phosphomimetic (T/E) and non-phosphomimetic (T/A) mutants to explore what effect phosphorylation of these residues might have on splicing. Transfection of these mutant constructs showed that Thr113 and Thr118 are critical in regulating the splicing activity of RBPMS-A, with the T/E mutant showing reduced activity. Furthermore, the RBPMS T/E mutant showed a decrease in nuclear localization which potentially could explain reduced splicing regulation. However, in vitro experiments with the recombinant mutated protein also showed reduced splicing regulation, independent of localization. Recombinant RBPMS-A proteins harboring these phosphomimetic mutations showed a significant reduction in splicing in vitro as well as a reduced RNA-binding and reduced higher-order oligomerization. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, I identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, these data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.

Peroxisomes are small, single membrane bound organelles with key roles in cellular lipid and hydrogen peroxide metabolism. They contribute to a wide range of metabolic processes including the β-oxidation of fatty acids and the synthesis of ether-phospholipids. To fulfil those functions, peroxisomes cooperate with other organelles, such as the endoplasmic reticulum (ER) and mitochondria. This collaboration requires close proximity of the organelles, which is mediated by protein tethering complexes that physically bridge apposing organelles. This thesis focuses on the interaction of peroxisomes with the ER that are mediated through interaction of the peroxisomal membrane protein ACBD5 and the ER-resident protein VAPB. These peroxisome-ER contacts are important for peroxisome motility, and the transfer of membrane lipids and ether-phospholipid intermediates. ACBD5 binds to VAPB via its FFAT motif. However, molecular mechanisms, which regulate formation of these contact sites, are unknown. Additionally, ACBD5 deficient patients present with neurological problems, but the pathological mechanisms are not well understood. Here, I explore the regulation of peroxisome-ER contacts and study them in model organisms. My findings reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. I show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT motif, which alter interaction with VAPB - and thus peroxisome-ER contact sites - differently. Moreover, I demonstrate that the kinase GSK3β regulates this interaction. These results reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of the FFAT-VAP interaction. In addition, I used two model systems, the fruit fly Drosophila melanogaster and the filamentous fungus Ustilago maydis, to study the impact of ACBD5 depletion on peroxisome dynamics in vivo in long polarized neurites and hyphae, respectively. I discovered that the D. melanogaster ACBD5 and VAP homologs interact, while the U. maydis proteins do not, but depletion of ACBD5 in both species leads to redistribution of peroxisomes. Overall, this provides valuable findings to increase our understanding of the pathophysiologic processes in ACBD5 deficient patients.

Parkinson's disease (PD) variants of the LRRK2 kinase increase the phosphorylation of its substrate Rab GTPases. Previous work indicated that Rab29, one of the LRRK2 substrates and a candidate gene for PD risk, might play an upstream role in LRRK2 kinase activity. It was shown that Rab29 overexpression in cells, together with LRRK2, enhances LRRK2 autophosphorylation and phosphorylation of its other substrate Rab GTPases. This effect is enhanced by the hyper-activating PD variants of LRRK2. At first, I focused on the Rab29-dependent activation. I identified the key role of the LRRK2 N-terminal armadillo and ankyrin domains as well as the GTP-bound state of LRRK2 in the activation. Our collaborators have shown that Rab29 overexpression recruits cytosolic LRRK2 to the Golgi, where Rab29 resides, and that the membrane-localisation is key for LRRK2 activation. Due to these findings, I focused further on LRRK2 localisation in response to overexpression of Rab29 and other Rab GTPases. This work revealed that Rab29, as well as Rab12 and two non-LRRK2 substrates Rab32 and Rab38, are able to recruit overexpressed LRRK2 via the N-terminus of LRRK2, while a group of other LRRK2 substrate Rab GTPases (Rab8a/b, Rab10, Rab35, Rab43) recruit LRRK2 independent of its N-terminus. I narrowed down the minimal N-terminal fragment of LRRK2 to a 150 amino acid region within the armadillo domain that is sufficient to be recruited by Rab29 and I investigated potential key residues necessary for the interaction. For another project, I investigated the role of 98 variants of LRRK2 identified in PD patients on LRRK2 cellular functions. Together with A. Kalogeropulou and Dr F. Tonelli, I evaluated the LRRK2 variant phosphorylation of endogenous Rab10 in cells and phosphorylation of LRRK2 biomarker sites. With the help of Dr Alan Prescott from the Dundee Imaging Facility I also evaluated the effect of the LRRK2 variants on recruitment to the Golgi by Rab29 and ability to localise to the microtubules under inhibitor treatment. Through this work we have identified multiple variants that were previously unknown to hyper-activate LRRK2, as well as some that decreased LRRK2 kinase activity in cells. None of the variants were able to disrupt LRRK2 co-localisation with Rab29, however, I have identified variants that disrupted microtubule localisation of LRRK2. My preliminary data has shown lack of a link between LRRK2 kinase activity in vivo and microtubule co-localisation and even though most of the known pathogenic variants of LRRK2 had high phosphorylation of Rab10, they had varied levels of association with microtubules suggesting that microtubule association is not a reliable marker for variant pathogenicity. For my final project, in collaboration with Prof. A. Khan's group I investigated the interaction between one of the LRRK2 substrates Rab8a and its phospho-specific interactor RILPL2. I have optimised an assay to produce large quantities of Rab8a phosphorylated at the LRRK2 site using a promiscuous kinase MST3. This phosphorylated Rab8a was used by Prof. A. Khan to determine the crystal structure of the complex of phosphorylated Rab8a and RILPL2 RH2 domain. I performed co-immunoprecipitation experiments using multiple mutants of RILPL2 RH2 domain to verify the key residues for complex formation. Together, this work has provided important knowledge that can be utilised for further study of the LRRK2 and Rab GTPase pathways - as well as to aid selection of PD patients with LRRK2 variants that would most likely benefit from LRRK2 inhibitor treatments in clinical trials.

Short linear motifs, made up of 2-10 amino acids in linear sequence space, are a central component of cellular decision making through proteins. They form a modular system in cells where combinations of domains and motifs are used as basic functional building blocks through interactions. Functions mediated through these motifs include cellular localisation, post-translational modifications, degradation and general protein-protein interactions. Since motifs are made up of a small number of amino acids they have unusual evolutionary properties, for instance they can evolve de novo, or be lost, through a small number of substitutions. This is of particular importance in pathogens such as viruses. Many viruses evolve new host-like motifs to interact with the host and change the regulation and signalling landscape within host cells to mediate infection. In this body of work, I have used influenza as a model to elucidate aspects of the evolutionary properties of motifs. I have been able to leverage recent progress made in determining nucleotide mutation rates and have developed a model for motif evolution that is defined from the nucleotide and codon levels. Simulations using this methodology suggested that different codons have varying propensities to evolve into amino acids within a linear motif. In other words, some sequences have higher motif evolvability. The simulations also indicated a fitness benefit to use some codons over others to encode linear motifs, due to the varying propensity to evolve. These findings suggest that motifs that are encoded by specific codons have higher motif evolutionary robustness, i.e. they can tolerate more mutations without affecting function. I went on to investigate if these predicted properties have played a role in motif evolution in influenza. I found that conserved motifs in influenza use the codons inferred to have higher evolutionary robustness. This would lead to increased fitness, as motifs are less often lost through mutations. I also found that this mutational robustness acts on stop codon usage in influenza, suggesting an explanation for an old observation of predominant use of TAA in many organisms. Interestingly, it also appears that evolutionary robustness of a motif can be varied to tune the rate of motif change, which influenza utilises in glycosylation motifs that interface with the host immune system. Finally, I investigated whether the codon choice and evolvability at early stages of viral host shifts could be used to predict the emergence of functional motifs. I have found that motif evolvability can aid the prediction of motif emergence. For influenza strains H1N1 and H3N2, which were introduced in the human population from birds during the 1900s, the sequence of the early strains could be used to predict the majority of the glycosylation sites that would emerge the following decades. The predictability of motif emergence could have important implications for vaccination efforts. The methodologies developed here, and the observations made about how motif evolution is shaped by codon choices in a predictable way will be important for a better understanding of the evolution of complexity and regulation involving motifs. This may have implications for complex diseases such as cancers, and for our understanding of the evolution of pathogen innovations and functionality.

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.

The burden of disease is a major issue in aquaculture production. The fish gill is a significant portal of entry for pathogens in fish. To investigate epithelial integrity and innate immune responses in gill epithelia the salmonid gill cell line RTgill-W1 was stimulated with diverse pro-inflammatory and infectious agents. Epithelial integrity in polarised cells expressed as transepithelial electrical resistance (TER) immediately increased after stimulation with different PAMPs (pathogen associated molecular patterns). In parallel tight junction and innate immune gene expression was modulated with bacterial PAMPs, LPS and MDP, internalized through actin dependent and independent endocytic pathways. The salmonid alphavirus subtype 2 (SAV-2) was used as an infectious agent in RTgill-W1 cells and was found to multiply at a low level. TER was found to be disturbed at an early stage of infection although tight junction related gene expression was not modulated. However, a strong PAMP-driven antiviral response was observed including upregulation of the expression of Rig-like receptor (RLRs) and several interferon stimulated genes (ISGs). Barrier function of trout gill epithelium against infection was also investigated. A bacterial invasion assay using A. salmonicida highlighted the capacity of PAMP pre-treatment to reduce bacterial invasion. At the level of signal transduction of the phosphoproteome of RTgill-W1 cells in steady state and under poly(I:C) and MDP stimulation was conducted where poly(I:C) stimulated cells presented a higher number of phosphoproteins (360 unique phosphoproteins). These results provide an untargeted view of the key signaling pathways that are rapidly activated in response to PAMP stimulation. The findings of the present study highlight an integrated response to viral infection and PAMP challenge in fish epithelia that could facilitate the development of novel therapeutic avenues for fish health management in aquaculture.

Proteins in nature are synthesized from a conservative set of 20 canonical amino acids, limiting the chemical space of biological systems. Over the last few decades, scientists have developed methods to expand the genetic code of living organisms, introducing new, non-canonical amino acids with diverse chemistries into proteins. These methods rely on engineering of the translational machinery often importing an aminoacyl-tRNA synthetase (aaRS)/tRNA pair that does not cross-react with the endogenous aaRS/tRNA pairs. This approach has allowed for co-translational incorporation of a variety of non-canonical amino acids, including amino acids for photocrosslinking, biophysical probes, amino acids bearing post-translational modifications or bio-orthogonal chemical handles for protein labelling and imaging. These designer amino acids allow researchers to probe, image and control protein function in vivo with great precision and minimal perturbation. Cultured mammalian cells present an attractive model for studying human biology and disease. The first aaRS/tRNA pair used for genetic code expansion in mammalian cells was the Escherichia coli (Ec) tyrosyl-tRNA synthetase (TyrRS) and Bacillus stearothermophilus (Bs) tyrosyl-tRNA (tRNATyr), however, this pair was quickly supplanted by Methanosarcina barkeri (Mb) and Methanosarcina mazei (Mm) pyrrolysyl-tRNA synthetase/tRNA pairs (PylRS/tRNAPyl), which are easily engineered to incorporate a wide variety of useful non-canonical amino acids. The developments in the genetic code expansion in mammalian cells have allowed researchers to study role of post-translational modifications, dissect signalling pathways, label and identify cell proteomes and identify protein-protein interactions directly in vivo. This thesis presents several key advances in, and applications of, genetic code expansion in mammalian cells. Chapter 1 introduces the relevant aspects of protein translation and summarises the progress in the field of genetic code expansion to date. Chapter 2 describes genetic encoding of phosphoserine and its non-hydrolyzable analogue in mammalian cells. The engineered phosphoseryl-tRNA synthetase/tRNA pair (SepRSv1.0/tRNAv1.0CUA) derived from Methanococcus mariplaudis and Methanocaldococcus janaschii (Mj) is shown to be orthogonal in mammalian cells. Subsequently, phosphoserine incorporation into a reporter protein is optimised by engineered translation elongation factor 1 alpha, engineered eukaryotic release factor 1 and metabolic engineering of the mammalian cell line. Overall this approach achieves an order of magnitude improvement in protein yield over unoptimised system. Incorporation of non-hydrolysable phosphonate analogue of phosphoserine in an engineered cell line is subsequently demonstrated and used for synthetic activation of a protein kinase. Chapter 3 describes two methodological advances in the use of genetic code expansion for protein imaging. Firstly, we demonstrate the use of genetic code expansion for super-resolution microscopy. Secondly, we identify aberrantly extended endogenous proteins ending with the amber stop codon as a source of non-specific labelling. We then proceed to minimise this background by optimisation of the labelling protocol and use the resulting protocol for live-cell imaging of a recently discovered microprotein. Chapter 4 experimentally demonstrates the orthogonality of a recently discovered PylRS/tRNAPyl pair from Methanomethylophilus alvus (Ma) in mammalian cells. We further demonstrate that this pair is mutually orthogonal to the widely used MmPylRS/tRNAPyl pair, establishing a new, orthogonal pair for genetic code expansion in mammalian cells. The two pairs are used to site-specifically direct two distinct amino acids into a reporter protein.

Cells are constantly sensing and adapting to changes in conditions. Protein post-translational regulation is one of the fastest mechanism used by cells to relay signals from sensors to effectors during such adaptations. Mass spectrometry allows for the study of posttranslational modifications on a very large scale and has been extensively applied to study protein phospho- rylation. On the order of 75% of human proteins have been estimated to be phosphorylated and approximately 160,000 human phosphosites are listed in public repositories. This wealth of knowledge, remains mostly uncharacterized with around 5% of human phosphosites having an annotated regulatory role or known regulatory kinase. Devising ways to study the functional importance of phosphosites is therefore a crucial research question. The recognition of target sites by a kinase is thought to be determined by a short contiguous sequence motif around the target phosphosite. It has been reported that kinases can, in some cases, recognize a 3D epitope instead of a linear peptide sequence. However, the extent by which 3D epitopes are important for kinase recognition is unknown. To study the usage of 3D kinase recognition motifs, I firstly examined if known in vitro and in vivo human kinase targets can be explained by 3D epitopes. For this I devised a computational pipeline mapping known kinase target phosphosites to structural models. Using these I identified potential cases where the important specificity determinant residues are not observed in contiguous sequences in the targets but may exist as a 3D epitope, and performed docking simulations to examine the possible kinase interactions. The 3D epitope examples were found to be rather exceptions than a rule, and the analysis confirms the general rule of linear motif recognition by kinases. To better predict phosphorylation of high functional relevance I analysed phosphosites that are highly conserved across species within protein domains families. These regions of conserved phosphorylation, defined as phosphorylation hotspots, were determined us- ing phosphosite data for a total of 40 eukaryotic species. A total of 241 domain regions were identified as hotspots within 162 domain families that were then mapped to proteins structures. These regions were shown to predict known regulatory sites and overlap with important structural features (i.e. protein interfaces and residues near or at catalytic sites). To further study the regulatory regions of protein domains I searched for regions of conserved ubiquitination and/or a high degree of recurrent mutations found in cancer. Of 68 domains that had enough data for analysis of all 3 types of hotspots I present the analysis of interesting cases and domains containing overlapping PTM and/or mutational hotspots.

Mutations in the E3 Ub ligase Parkin (PARK2) and the protein kinase PINK1 (PARK6) are genetically linked to Young-Onset Parkinson's Disease. The two enzymes orchestrate clearance of damaged, cytotoxic mitochondria through a specialized form of autophagy termed mitophagy. By phosphorylating ubiquitin (Ub) on the surface of damaged mitochondria, PINK1 generates a phospho-Ub receptor for autoinhibited cytosolic Parkin. Once localized to the mitochondrial surface, Parkin is activated by direct PINK1-mediated phosphorylation on its Ub-like domain (Ubl). To efficiently recruit the autophagy machinery, activated Parkin transfers Ub from upstream E2 Ub-conjugating enzymes to substrates on the mitochondrial surface via an E3~Ub intermediate. Resolving the precise mechanism of Parkin activation by PINK1-mediated phosphorylation is the primary aim of this thesis. Additionally, building on previous work in which a novel phospho-Ub conformer was characterized crystallographically, the same conformer was found in unmodified Ub in this work. This striking finding is supported by further biochemical and biophysical investigation of the newly discovered equilibrium in unmodified Ub. To address the mechanism of Parkin activation and open new avenues for translational research, both dynamic and crystallographic approaches were employed. Hydrogen-deuterium exchange mass spectrometry reveals that Parkin phosphorylation enables a conformational equilibrium between an autoinhibited and an active Parkin state. In the active state the phospho-Ubl domain binds to the Unique Parkin domain (UPD) of the Parkin core and thereby displaces the catalytic RING2 domain from its autoinhibitory position. Catalytic intermediates, such as the E2-bound state or the Ub-charged Parkin species shift the equilibrium in favour of the active conformer. Parkin-mediated substrate ubiquitination occurs by a flexibly tethered catalytic RING2 domain independently of the Parkin core, in line with its minimal substrate selectivity observed in vitro. The new activating interface between the Parkin phospho-Ubl and the Parkin core is revealed in a 1.8Å crystal structure of phosphorylated human Parkin lacking the flexible catalytic domain. Additionally to the phospho-Ubl, a conserved linker region, the activating element (ACT) aids displacement of the catalytic RING2 domain by mimicking RING2 autoinhibitory interactions with the Parkin core. This crystal structure explains patient mutations in the UPD phosphate-binding pocket as well as in the newly identified ACT. Together, the clinically relevant molecular insights described in this thesis may facilitate the development of therapeutic or diagnostic tools for Parkinson's disease.

Translational control is a powerful means to alter gene expression and regulates synaptic plasticity, learning and memory. 4E-BP2 (Eif4ebp2, Eukaryotic Initiation Factor 4E-Binding Protein 2) is the predominantly expressed 4E-BP in the mammalian brain and represses cap-dependent translation initiation, by binding to eIF4E (Eif4e, eukaryotic Initiation Factor 4E). As a master regulator of protein synthesis in the mammalian brain, 4E-BP2 has been implicated in learning, memory and Autism Spectrum Disorder (ASD). Upon phosphorylation by mTOR (mammalian Target Of Rapamycin, mTOR) which occurs in most tissues, 4E-BP2 cannot bind to eIF4E, failing to repress translation initiation. However, in early postnatal brain development, 4E-BP2 undergoes brain-specific post-translational deamidation on asparagines N99 and N102, which are converted to aspartic acid. Asparagine deamidation is not catalysed by enzymes but can occur spontaneously and is induced by alkaline pH. Deamidated 4E-BP2 was shown to regulate the kinetics of excitatory synaptic transmission in early postnatal brain development, suggesting that it may be important for synaptic function during that crucial developmental period. N99/N102 deamidation decreases the affinity of 4E-BP2 for eIF4E and increases its binding to the mTORC1 protein Raptor. The significance of enhanced Raptor binding to deamidated 4E-BP2 is yet unclear because 4E-BP2 phosphorylation is very low in adult brain. Moreover, the role of deamidated 4E-BP2 and the downstream effects of deamidated 4E-BP2 translational control in the mammalian brain are not known but of cardinal importance given the pervasive role of 4E-BP2 in regulating brain function. In this thesis, we describe a previously unidentified mechanism during early postnatal brain development, whereby the constitutively deamidated form of the cardinal brain translation initiation repressor 4E-BP2 is more susceptible to ubiquitin proteasomal degradation (as compared to unmodified, WT protein) because it binds with higher affinity to a complex, comprising the mTORC1 protein Raptor and the ubiquitin E3 ligase CUL4B. Deamidated 4E-BP2 (2D) stability is regulated by mTORC1 and AMPAR activity but not NMDARs. We also showed that 4E-BP2 deamidation is neuron-specific and occurs in human brain. We explored whether deamidated and WT 4E-BP2 have a similar subcellular distribution in neurons, indicating that there is very low co-localization of them in both soma and dendrites. We studied WT and 2D structures, with Synchrotron radiation circular dichroism (SCRD), Small angle X-ray scattering (SAXS) and Nuclear magnetic resonance (NMR) spectroscopy of full-length recombinant 4E-BP2 (WT or 2D) expressed in E. Coli and purified, and we identified that they share a similar structure, with only minor differences in a few residues. Moreover, using unbiased translatome mapping, we discovered that overexpression of deamidated 4E-BP2 represses the translation of a distinct pool of mRNAs linked to cerebral development, mitochondria and chiefly NF- κB activity. Collectively, these data describe a previously unidentified brain-specific translational control mechanism that could be crucial for postnatal brain development in neurodevelopmental disorders such as ASD.

In eukaryotes, cell proliferation and cell cycle transitions are strictly controlled by the anaphase-promoting complex/cyclosome (APC/C). The APC/C is an E3 ubiquitin ligase that regulates chromatid segregation at the metaphase to anaphase transition, exit from mitosis and the establishment and maintenance of G1. The APC/C’s catalytic activity and substrate specificity are controlled by its interactions with two coactivators, Cdc20 and Cdh1. In contrast to Cdh1, APC/C activation by Cdc20 during mitosis requires hyper-phosphorylation of APC/C subunits by cyclin-dependent kinase (Cdk) and polo kinase. The aim of the first part of this thesis was to understand how mitotic phosphorylation regulates APC/C activity. Using cryo-electron microscopy and biochemical analysis, we found that an auto-inhibitory segment of the Apc1 subunit acts as a molecular switch that in apo unphosphorylated APC/C interacts with a coactivator-binding site (C-box binding site), thereby obstructing engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box binding site to relieve APC/C auto-inhibition. Efficient phosphorylation of the auto-inhibitory segment requires the recruitment of the kinase Cdk-cyclin-Cks to a hyper-phosphorylated loop of Apc3. In addition to regulation of APC/C activity by phosphorylation, ordered cell progression is ensured by the ability of the APC/C to target substrate degradation in a defined order. At mitosis onset, degradation of securin and cyclin B1 is inhibited by the spindle assembly checkpoint, exerted by the mitotic checkpoint complex (MCC), whereas both cyclin A2 and Nek2A are not subject to MCC inhibition. The aim of the second part of the thesis was to elucidate the mechanism of how the APC/C achieves its substrate specificity. Our biochemical analysis showed that the resistance of cyclin A2 to MCC inhibition is due to its ABBA motif and the Cdk-associated Cks2 subunit. Furthermore, we found that it is the Cdc20 molecule of the MCC that binds to the ABBA motif to allow for cyclin A2 ubiquitination. Strikingly, mutating all three known degrons (KEN box, D box and ABBA motif) of cyclin A did not affect its ubiquitination by APC/CCdc20. Deletion of a potential novel degron found within residues 60-80 of cyclin A2 impaired cyclin A2 ubiquitination.

Aurora A and B are the major kinases that control key events in mitosis, such as centrosome function, spindle assembly, chromosome segregation and cytokinesis, through phosphorylation of multiple proteins. These kinases share identical consensus target motifs, so the substrate specificity is determined by distinctive sub-cellular localization of the Auroras. Many proteins have been identified as targets of either Aurora A, or Aurora B, or both kinases by mass spectrometry studies. However, only a few of the identified phosphorylation sites in these targets have a characterized function in vivo. Therefore, the molecular mechanisms underlying the regulation of certain mitotic events by Aurora kinases remain unclear. The objective of my work was to develop a tool for identifying new substrates of both Aurora kinases. More specifically, I aimed to identify the molecular targets of Aurora A at the kinetochores, and determine how Aurora A contributes to the error correction near spindle poles. I first demonstrated that the outer kinetochore protein HEC1/Ndc80, phosphorylated by Aurora B at kinetochores, can also be phosphorylated by Aurora A close to the centrosomes (Chapter 2). My finding showed that Aurora kinases can share substrates in the cells and revealed the mechanism by which Aurora A contributes to the error-correction near spindle poles. To identify and characterise novel substrates of Aurora kinases, I developed a bioinformatic approach in collaboration with the Centre Bioinformatician, Alastair Kerr. This bioinformatic method uses the Auroras’ shared consensus motifs combined with several parameters that control the substrate specificity of Aurora kinases. I tested the phosphorylation of the chosen candidates in vitro using radiolabelled kinase assays. In my study, five proteins were validated - SPICE1, TTLL4, AHCTF1, CLASP2 and an uncharacterized protein KIAA1468 - as in vitro substrates of Aurora A and Aurora B kinases (Chapter 3). I then focussed on the Aurora kinases-dependent regulation of spindle and centriole-associated protein, SPICE1, in cells (Chapter 4). Using either site-directed mutagenesis of SPICE1 or inhibition of Aurora kinases with small molecule inhibitors, I found that the predicted phosphorylation of the SPICE1 C terminus had the function in cells of directing the SPICE1 localization on the spindle MTs. My results demonstrate the high accuracy of this genome-wide bioinformatics approach. By complementing mass spectrometry studies, here lies a potential for the identification of other unknown substrates, which is important for the general understanding of how Aurora kinases regulate the mitotic apparatus.

The tumour suppressor p53 is often disrupted by missense mutations that can result in p53 protein accumulation and acquisition of novel oncogenic activities. Various studies have demonstrated that DNA-damaging drugs currently used in the clinic aimed at activating wild type p53, can also stabilise and activate mutant p53 oncogenic functions and thereby paradoxically enhance tumour progression, resulting in poor response to the treatment. In this study we aimed to investigate whether, like in wt p53, post-translational modifications (PTMs) drive such drug-induced mutant p53 accumulation and activation. For this purpose, we generated plasmids expressing non-phosphorylatable and phospho-mimic versions of R175H mutant p53 and tested them in different cell line models. We demonstrated that in response to DNA damage mutant p53 is accumulated and phosphorylated and these phenomena appeared to be mediated by ATM and ATR kinases. DNA-damage induced acetylation was also observed and occurred in a S15 phosphorylation-dependent manner. This suggested a role of the HAT p300, which is recruited by phosphorylated S15. Of note, other works have shown that p300 is required to trigger some oncogenic functions of mutant p53. We then aimed at developing systems to explore mutant p53 functions and their dependence on PTMs. Although we showed that cell growth is compromised upon endogenous mutant p53 depletion, exogenous expression of mutant p53 or its phosphorylation-site forms did not result in a successful rescue in our experimental conditions, thus we were unable to use this strategy to test the effect of PTMs. Ectopic expression of R175H mutant p53 or its phosphorylayion-site versions did not interfere with the growth rate and response to chemotherapy of the p53-null cell line H1299. We also found that mutant p53 phosphorylation does not affect subcellular localisation of mutant p53 and mutant p53-mediated inhibition of p63. Interestingly, ectopically expressed mutant p53 enhanced cell migration in H1299 cells. Notably, our results suggested an apparent threshold effect of mutant p53 levels required to induce migration. Due to the difficulty of obtaining cell lines expressing similar levels of the different phosphorylation-site mutants, the determination of the role of phosphorylation in mutant p53-induced migration was not conclusive. Remarkably, we found that, while S15 and S20 phosphorylation decreased MDM2-dependent degradation, only phosphorylated S20 interfered with CHIP-induced turnover in H1299 cells. Overall our data suggest that, despite exhibiting opposite biological effects, mutant and wt p53 can share upstream regulatory mechanisms and thus present phosphorylation as a promising target to prevent mutant p53 stabilisation and activation and improve response to therapy. Our results also highlight the challenge of developing a good system for determining the effects of the mutant p53 protein and its regulation by PTMs.

A plant’s survival is largely impacted by its ability to sense and respond to gravitational changes, such as the changes that occur in spaceflight. However, the gravitropic signaling pathway is not fully understood. In the BRIC-20 experiment aboard the International Space Station, the Arabidopsis proton pump AHA2 was found to be differentially phosphorylated in microgravity compared to ground controls. AHA2 is hypothesized to be involved between plant hormone movement and differential growth during gravity signaling. When subject to reorientation, aha2 mutant seedlings had increased root curvature compared to wild type. Furthermore, wild type seedlings treated with fusicoccin, a phytotoxin that increases AHA2 phosphorylation and activity, had increased root curvature compared to untreated plants. To determine the role of AHA2 phosphorylation in gravitropic signaling, several lines of transgenic Arabidopsis with modifications in phosphorylation sites are being developed. The transformants will then be phenotyped for altered gravity response, showing if the phosphorylation of the AHA2 protein is involved in a plant’s gravity response. These findings will further knowledge on the molecular mechanisms of gravitropism to develop space-tolerant plants for life support in spaceflight and for habitation of the moon, Mars, and beyond.

The PI3K/AKT signaling pathway is frequently dysregulated in cancer and controls critical cellular processes such as survival, proliferation, metabolism and growth. Additionally, protein glycosylation is essential for proper protein folding and is also frequently deregulated in cancer. Cancer cells require increased protein folding capacity to sustain high proliferation rates. The glycosyltransferase alpha-1,3-mannosyltransferase (ALG3) catalyzes the addition of mannose units to an N-glycan precursor during glycan biosynthesis. ALG3 is a rate-limiting enzyme during glycan biosynthesis in the endoplasmic reticulum. Using in vitro approaches and cell-based assays, we discovered that in breast cancer cells ALG3 functions downstream of the PI3K pathway, and is directly phosphorylated by AKT at Ser11/Ser13. This represents a direct link between PI3K/AKT oncogenic signaling and protein glycosylation. Additionally, we found that the ALG3 gene resides proximal to the PIK3CA gene in the 3q26 amplicon. Consequently, PIK3CA and ALG3 are co-amplified in both lung, breast and ovarian carcinomas. Using cell-based assays, we found that depletion of ALG3 leads to improper glycan formation, induces ER stress and reduces proliferation of breast cancer cells. We further used lectin staining approaches to reveal the functional consequence of PI3K/AKT-mediated regulation of ALG3 function, including the proper folding and surface expression of receptor tyrosine kinases. The studies outlined in this thesis support a model in which in normal cell physiology, growth factor stimulation of the PI3K/AKT pathway leads to phosphorylation of ALG3 at Ser11/Ser13 to facilitate the rate of protein N-glycosylation. Similarly, we further propose a model in which the demands of a rapidly proliferating cancer cell are in part mediated by increased proper protein folding facilitated by ALG3 regulation by hyperactive PI3K/AKT. Consequently, depletion of ALG3 leads to improper glycan formation, proteins misfolding and induction of ER stress and the unfolded protein response. Collectively, these findings advance our understanding of the regulation of N-glycosylation by oncogene-driven signaling and its role in cancer progression, and in turn pave the way for exploring future combination strategies targeting PI3K/AKT and protein glycosylation.

Mitosis is a highly regulated process by which a cell duplicates and distributes its chromosomal DNA into two identical daughter cells equally. Equal distribution of the chromosomes is crucial for accurate propagation of genetic information. This is essential for maintaining viability and preventing genomic instability that can potentially lead to cancer. In order to avoid unequal distribution of chromosomes, cells employ a surveillance mechanism called the spindle assembly checkpoint (SAC). The SAC is an inhibitory signalling network, which delays segregation of chromosomes, until they have stably attached to spindle microtubules through their multi-protein platforms, known as kinetochores. The main target of the SAC is the anaphase promoter complex/ cyclosome (APC/C), an E3 ubiquitin ligase. Specifically APC/C and its activator Cdc20 are inhibited by the main effector of the SAC, called the mitotic checkpoint complex (MCC). The MCC consists of Cdc20, Mad2, Mad3 and Bub3 (except S. pombe) proteins, which are recruited to the unattached kinetochores to promote MCC assembly. Once the chromosomes stably attach to the spindle, the SAC is turned off, MCC disassembles, and APC/CCdc20 is released from the inhibition. Activated APC/CCdc20 then targets its two main substrates, securin and cyclin B, for proteasomal degradation, and thereby triggers anaphase onset and mitotic exit. SAC signalling involves many protein components, whose activities are essentially regulated by direct protein-protein interactions and/ or post-translational modifications. One of these major modifications is phosphorylation, which is mediated by the SAC kinases such as Aurora B, Mps1 and Bub1. A number of studies have characterised SAC related substrates of Aurora B and Mps1 kinases in several model organisms. On the other hand, Bub1 kinase activity has been thought to play a key role in chromosome bi-orientation and more of an auxiliary role in SAC activation. The aim of this study is to investigate the importance of Bub1 kinase activity for SAC response in fission yeast Schizosaccharomyces pombe (S. pombe). SAC activation assays, using various degrees of spindle perturbation, have demonstrated that Bub1 kinase activity plays an important role in SAC maintenance. In order to examine the pathways downstream of Bub1, we set out to indicate Bub1 substrates which may be involved in SAC signalling. According to studies in various species, Cdc20 appears to be a prominent candidate, whose phosphorylation by Cdk1 and Bub1 kinases has been reported to regulate its mitotic activity. To investigate whether Cdc20 is phosphorylated by Bub1 in vitro, we purified recombinant S. pombe proteins from insect cells. Subsequent kinase assays identified Cdc20 as an in vitro substrate of Bub1, and the phosphorylated sites in Cdc20 were mapped by mass spectrometry. To address if this phospho-modification is involved in SAC regulation, phosphorylation mutants of Cdc20 were analysed in terms of their abilities to activate and silence SAC in vivo. Results show that phosphorylation of Cdc20 C-terminus promotes SAC maintenance in response to spindle damage. Furthermore, the mutations mimicking Bub1-mediated phosphorylation of Cdc20 Cterminus restore the SAC defects in the absence of Bub1 kinase activity. In addition, we purified S. pombe mitotic checkpoint complex (MCC) from insect cells, and analysed the interactions between its components (Cdc20, Mad2 and Mad3) by cross-linking mass spectrometry. Crystal structure of S.pombe MCC has been determined recently, which lacks Mad3 C-terminus and flexible C-terminal tail of Cdc20. Using an MCC with full length Mad3, we identified novel interactions between the C-terminal tails Mad3 and Cdc20, which are in close proximity to the identified Cdc20 phosphorylation sites. Briefly, in this study we confirm the previously known roles of Bub1 kinase activity (chromosome bi-orientation). Moreover, we propose a new pathway (in addition to the well-established H2A pathway) mediated by Cdc20, that may be important to maintain the SAC response.

In this thesis, I investigate dynamic and computational properties of prokaryotic signalling architectures commonly known as the Two Component Signalling networks and phosphorelays. The aim of this study is to understand the information processing capabilities of different prokaryotic signalling architectures by examining the dynamics they exhibit. I present original investigations into the dynamics of different phosphorelay architectures and identify network architectures that include a commonly found four step phosphorelay architecture with a capacity for tuning its steady state output to implement different signal-response behaviours viz. sigmoidal and hyperbolic response. Biologically, this tuning can be implemented through physiological processes like regulating total protein concentrations (e.g. via transcriptional regulation or feedback), altering reaction rate constants through binding of auxiliary proteins on relay components, or by regulating bi-functional activity in relays which are mediated by bifunctional histidine kinases. This study explores the importance of different biochemical arrangements of signalling networks and their corresponding response dynamics. Following investigations into the significance of various biochemical reactions and topological variants of a four step relay architecture, I explore the effects of having different types of proteins in signalling networks. I show how multi-domain proteins in a phosphorelay architecture with multiple phosphotransfer steps occurring on the same protein can exhibit multistability through a combination of double negative and positive feedback loops. I derive a minimal multistable (core) architecture and show how component sharing amongst networks containing this multistable core can implement computational logic (like AND, OR and ADDER functions) that allows cells to integrate multiple inputs and compute an appropriate response. I examine the genomic distribution of single and multi domain kinases and annotate their partner response regulator proteins across prokaryotic genomes to find the biological significance of dynamics that these networks embed and the processes they regulate in a cell. I extract data from a prokaryotic two component protein database and take a sequence based functional annotation approach to identify the process, function and localisation of different response regulators as signalling partners in these networks. In summary, work presented in this thesis explores the dynamic and computational properties of different prokaryotic signalling networks and uses them to draw an insight into the biological significance of multidomain sensor kinases in living cells. The thesis concludes with a discussion on how this understanding of the dynamic and computational properties of prokaryotic signalling networks can be used to design synthetic circuits involving different proteins comprising two component and phosphorelay architectures.

Presently, progress in the field of phosphoproteomics has been accelerated by mass spectrometry. This is not a surprise owing to not only the accuracy, precision and high-throughput capabilities of MS but also due to the support it receives from informaticians whom allow the automated analysis; making the task of going from a complex sample to a statistically satisfactory set of phosphopeptides and corresponding site positions with relative ease. However, the process of identifying and subsequently pinpointing the phosphorylation moiety is not straightforward and remains a challenging task. Furthermore, it has been suggested that not all phosphorylation sites are of equal functional importance, to the extent that some may even lack function altogether. Clearly, such sites will confound the efforts towards functional characterisation. The work in this thesis is aimed at these two issues; accurate site localisation and functional annotation. To address the first issue, I adopt a multi-tool approach for identification and site localisation; utilising the different underlying algorithms of each tool and thereby allowing an orthogonal perspective on the same tandem mass spectra. Doing so enhanced accuracy over any single tool by itself. The power of this multi-tool approach stemmed from its ability to not predict more true positives but rather by removal of false positives. For the second issue, I first investigated the hypothesis that those of functional consequence exhibit stronger phosphorylation-characteristic features such as the degree of conservation and disorder. Indeed, it was found that some features were enriched for the functional group. More surprisingly, there were also some that were enriched for the less-functional; suggesting their incorporation into a prediction algorithm would hinder functional prediction. With this in mind, I train and optimise several machine-learning algorithms, using different combinations of features in an attempt to (separately) improve general phosphorylation and functional prediction.

In fission yeast, microtubule (MT) nucleation is regulated in space and time. In interphase, MTs are nucleated in the cytoplasm to regulate cell polarity, whereas in mitosis, nucleation takes place inside the nucleus to form a mitotic spindle. We hypothesize that several non-exclusive mechanisms may be responsible for this differential regulation of MT nucleation. Two fission yeast proteins, Mto1 and Pcp1, are involved in MT nucleation in interphase and mitosis, respectively. These proteins share a sequence motif, called CM1 that is responsible for interaction with the γ-tubulin complex (γ-TuC). In the first part of my project, I tested whether sequence differences between Mto1 and Pcp1 CM1 regions contribute to the differential regulation of MT nucleation in interphase vs. mitosis. I showed that the two CM1 regions are interchangeable and play no role in differential regulation of Mto1 and Pcp1. By generating Pcp1-9A1 mutant, where conserved residues within the Pcp1 CM1 region was replaced with alanines, I showed that Pcp1 CM1 region is required for its function. Moreover, using CM1 regions from two human proteins that are implicated in schizophrenia and microcephaly development, MMGL and CDK5RAP2, I showed that human CM1 domains could rescue yeast protein function, demonstrating that the CM1 region is conserved across evolution. In the second part of my project, I focused on regulation of cytoplasmic MT nucleation. In fission yeast, cytoplasmic MT nucleation occurs from several distinct sites in the cell and is promoted by the Mto1/2 complex. The Mto1/2 complex is composed of multiple copies of Mto1 and Mto2 and interacts with the γ-TuC. Disruption of the interaction of Mto1/2 with the γ-TuC, or of the Mto1-Mto2 interaction, results in a complete loss of interphase cytoplasmic nucleation. As cells enter mitosis, Mto2 is hyperphosphorylated, and the Mto1-Mto2 interaction is disrupted, leading to abolishment of cytoplasmic nucleation. This led to a hypothesis that Mto2 phosphorylation regulated the Mto1/2 complex mitotic disassembly. I showed that Mto2 phosphorylation is used to control levels of cytoplasmic nucleation in both interphase and mitosis. During interphase, I found that Mto2 is phosphorylated in order to reduce levels of MT nucleation. When Mto2 phosphorylation is prevented by mutation of phosphorylatable residues to alanines, Mto1/2 mutant complexes show a more robust interaction with the γ-TuC, and more MTs are nucleated in the cytoplasm. During mitosis, hyperphosphorylation of Mto2 plays a role in the disassembly of Mto1/2 complexes. In particular, while the interaction of wild-type Mto2 with Mto1 is disrupted during mitosis, Mto2-alanine mutants, in which phosphorylation was nearly abolished, are still able to interact with Mto1 in mitosis. Interestingly, Mto1/2 complexes containing Mto2-alanine mutants are still disassembled in mitosis by disruption of Mto2 self-interaction. I used SILAC phosphoproteomics to show that Mto2-alanine is still phosphorylated in mitosis, suggesting the Mto2 self-interaction might also be controlled by phosphorylation. While doing so, I developed a novel SILAC quantification method that is particularly useful for quantification of multiply phosphorylated proteins and peptides. Using data obtained by SILAC, I generated additional Mto2 alanine mutants with more phosphorylation sites mutated. Preliminary analysis showed that these mutants are similar to the alanine mutants analysed previously; however, more analysis is required to generate more definitive conclusions. In summary, in this study I have uncovered the functional conservation of the CM1 region from yeast to human. I also showed that Mto2 phosphorylation regulates cytoplasmic MT nucleation in both interphase and mitosis, by regulating the Mto2-Mto1 interaction and the Mto2-Mto2 self-interaction and therefore remodelling the Mto1/2 complex.

In mammals, there are two isoforms of eukaryotic translation elongation factor 1A (eEF1A) called eEF1A1 and eEF1A2. They share 98% similarity at the amino acid level, and the main function of both is to facilitate the elongation process in protein translation. However, they have very different expression patterns. While eEF1A1 is universally expressed, eEF1A2 is strictly expressed in muscle, brain and heart. The over-expression of eEF1A2 has been found in cancers, such as ovarian and breast cancer. The factors influencing the different expression patterns of the two isoforms and the mechanisms by which eEF1A2 can act as an oncogene are not clear, therefore, the main aim of this study was to further investigate these two areas. The first aim was to find out whether the resveratrol induced down-regulation of eEF1A2 was mediated by miR-663. Western blotting in MCF7 cells showed that the level of endogenous eEF1A2 was decreased after resveratrol treatment while eEF1A1 remained stable. In contrast, NIH-3T3 stable cell lines which stably express the eEF1A2 coding sequence (CDS) only did not show this down-regulation, suggesting that the untranslated regions (UTRs) might play a role in this regulation. I then showed that miR-663 has ability to down-regulate a reporter linked to the UTRs of eEF1A2. The same reporter gene harbouring UTRs in which the binding sites of miR-663 had been deleted also showed down-regulation after resveratrol treatment, suggesting that the UTRs of EEF1A2 are key to the down-regulation of eEF1A2 by resveratrol but that miR-663 does not mediate this decrease. The second project aimed to address why eEF1A2 is an oncogene but eEF1A1 is not. The 3D structure of human eEF1A1 and eEF1A2 shows that the most of the highly conserved amino acids differences between the two isoforms are Ser and Thr residues, which are potential sites for phosphorylation. I mutated these three sites in eEF1A2 expression constructs to the equivalent amino acid from eEF1A1. Firstly, by transient transfection, all the mutant eEF1A2 were shown expressed and the sub-cellular locations of eEF1A2 remain unchanged after site-directed mutagenesis. Then, stable cell lines were generated. Anchorage independent growth (soft agar) and focus formation assays showed that the stable cell lines harbouring wild type eEF1A2 were significantly more transformed that those expressing the eEF1A2 mutants. However, there was no apparent difference in global protein synthesis between these cell lines. The results suggest that the potential phosphorylated sites in eEF1A2 play an important role in its oncogenicity and that this oncogenicity is not related to the canonical function of eEF1A2.

The activity of protein kinases is heavily dependent on the phosphorylation state of the protein. Kinase phosphorylation states have been prepared through biological or enzymatic means for biochemical evaluation, but the use of protein chemical modification as an investigative tool has not been addressed. By chemically reacting a genetically encoded cysteine, phosphocysteine was installed via dehydroalanine as a reactive intermediate. The installed phosphocysteine was intended as a surrogate to the naturally occurring phosphothreonine or phosphoserine of a phosphorylated protein kinase. Two model protein kinases were investigated on: MEK1 and p38α. The development of suitable protein variants and suitable reaction conditions on these two proteins is discussed in turn and in detail, resulting in p38α-pCys180 and MEK1-pCys222. Designed to be mimics of the naturally occurring p38α-pThr180 and MEK1-pSer222, these two chemically modified proteins were studied for their biological function. The core biological studies entailed the determination of enzymatic activity of both modified proteins, and included the necessary controls against their active counterparts. In addition, the studies on p38α-pCys180 also included a more detailed quantification of enzymatic activity, and the behaviour of this modified protein against known inhibitors of p38α was also investigated. Both modified proteins were shown to be enzymatically active and behave similarly to corresponding active species. The adaptation of mass spectrometry methods to handle the majority of project's analytical requirements, from monitoring chemical transformations to following enzyme kinetics was instrumental in making these studies feasible. The details of these technical developments are interwoven into the scientific discussion. Also included in this thesis is an introduction to the mechanism and function of protein kinases, and on the protein chemistry methods employed. The work is concluded with a projection of implications that this protein chemical modification technique has on kinase biomedical research.

The post-translational modification of proteins is a fundamental means of biological information processing, with important functions in development, homeostasis and disease. Post-translational modifications (PTMs) can dynamically diversify the proteome in response to intracellular and extracellular signals. Since thousands of modified residues as well as entirely new modification types have recently been discovered in proteins, elucidating their biological functions and identifying the protein components of these PTM systems is a fundamental problem. Chapter 1 gives an overview of the types and known biological functions of different PTMs, as well as experimental methods used to detect them. Intrinsic disorder in proteins is introduced as a structural feature which may influence local evolutionary rates. Several examples of complex PTM signalling systems are then described. Chapter 2 presents a study of the evolution of modified amino acids in human proteins. By analysing sequence, polymorphism and mutation data at the species, population and individual levels, we observed significant evolutionary constraints on all PTM types for which extensive data was available, as well as overrepresentation of amino acids which mimic modified residues at equivalent positions. Chapter 3 applies a framework for the identification of important components of PTM signalling systems to lysine acetylation. The proteins of this system were found to be similarly conserved as essential genes. Their evolutionary histories suggested a conserved origin in chromatin regulation, followed by functional diversification. Chapter 4 extends the scope to signalling via transcriptional regulation, and presents a comprehensive overview of the interactome of the stem cell transcription factor Oct4. The results presented here facilitated characterisation of a novel post-translational modifier of Oct4, the glycosyltransferase Ogt. Chapter 5 highlights my key findings from applying evolutionary and data integration approaches to signalling networks, and outlines their implications for the study of novel signalling systems and for their engineering in synthetic biology. This dissertation therefore illuminates evolutionary, structural and functional principles of cellular signalling networks across species and within populations.

The N-7 methylguanosine cap structure is conserved from yeast to man. It is essential for cell proliferation as it influences several steps in eukaryotic gene expression including transcription, pre-mRNA processing, RNA export and translation. The N-7 methylguanosine cap is added co-transcriptionally to RNA pol II transcripts. In mammals, two enzymes catalyse the synthesis of the N7-methylguanosince cap. RNGTT adds an inverted guanosine group to the first transcribed nucleotide and RNMT methylates the guanosine cap at the N7-position. RNMT consists of a catalytic domain and an N-terminal domain that is absent in lower eukaryotes. Experiments presented in this thesis revealed that the N-terminus mediates RNMT recruitment to transcription start sites. Furthermore, it was found that the RNMT N-terminal domain is phosphorylated at Threonine-77 (T77) by CDK1/Cyclin B in a cell cycle-dependent manner during G2/M-phase. RNMT T77 phosphorylation activates cap methyltransferase activity in vitro. Furthermore, it negatively regulates the interaction of RNMT with KPNA2 (Importin-a), which was found to inhibit RNMT activity in vitro. RNMT T77 phosphorylation is required for normal cell proliferation suggesting an important biological function. Initial experiments indicated that RNMT T77 phosphorylation functions to regulate gene expression in a gene-specific manner. Future work is focused on establishing an experimental system to perform a genome-wide study in order to elucidate which transcripts are affected by RNMT T77 phosphorylation. To summarise, this study for the first time revealed that the RNA cap methyltransferase activity is regulated in a cell-cycle dependent manner.

Fusel alcohols signal nitrogen scarcity to elicit a range of responses in the yeast Saccharomyces cerevisiae. These alcohols activate pseudohyphal growth and cause rapid inhibition of translation initiation. Previous work from our lab has highlighted that the translation initiation factor eIF2B is a target for this regulation. eIF2B is the guanine nucleotide exchange factor required for recycling eIF2•GDP to eIF2•GTP. The GTP bound form of eIF2 can interact with the Methionyl initiator tRNA to form the ternary complex. Fusel alcohols target eIF2B leading to reduced ternary complex; however the mechanism by which alcohols cause this effect is currently unknown. This study aims to characterize the effects of fusel alcohols on eIF2B and identify post-translational modifications, which may be responsible for translation inhibition. Following purification of eIF2B, a number of novel phosphorylation sites have been identified using mass spectrometry. In particular, phosphorylated serine has been identified at position 131 within yeast eIF2Bδ. Phosphoantibody analysis suggests that the phosphorylation status of this residue differs following fusel alcohol treatment. Mutagenesis experiments are consistent with phosphorylation of this residue being essential for the translational inhibition seen following fusel alcohol exposure. Therefore, phosphorylation of this residue may prime eIF2B for regulation and provide a switch to sensitize the process of translation to particular conditions.

The ability to sense and respond to environmental cues is crucial for the survival of all organisms. This response is often manifested by exerting control at different levels of gene expression, i.e. transcription, translation and post translation levels. Global control of protein synthesis is frequently exercised at the initial step of translation initiation and is generally achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them. The formation of ternary complex (TC) is considered first step of translation initiation and depends on the recycling of inactive eIF2-GDP to active eIF2-GTP form. This nucleotide exchange reaction is catalyzed by the eukaryotic initiation factor-2B (eIF2B). eIF2B is composed of a regulatory sub-complex of alphaβdelta subunits and a catalytic sub-complex of the γε subunits. The guanine nucleotide exchange activity of eIF2B is regulated by phosphorylation of eIF2alpha and additionally in mammalian cells, by direct phosphorylation of eIF2B at multiple sites in ε subunit, where most of the catalytic activity of eIF2B resides. Recent unpublished studies in the Pavitt laboratory identified novel phosphorylation sites by Mass Spectrometry in γ and ε subunits of eIF2B catalytic sub-complex. In order to study the functional significance of these phospho-sites for translation initiation, Site Directed Mutagenesis (SDM) was performed to generate Ser to Ala mutants. All mutations are viable and have no significant growth defect on rich or minimal media; however the significance of these sites in yeast growth became apparent by growing yeast in different stress conditions (e.g. Rapamycin, Torin1, amino acid starvation and 1-butanol). Effects on the phosphorylation pattern at these sites were monitored by using custom generated phospho-specific antibodies. All phosphorylation events appear independent of the eIF2alpha kinase (Gcn2p in yeast). The phosphorylation of ε-S528 depends on the presence of ε-S525. This study finds that addition of rapamycin, Torin1, amino acid starvation and butanol, which each inhibits global translation initiation, alters the phosphorylation pattern at ε-S435, ε-S525 and ε-S528 sites. Linking growth to phosphorylation, it appears that phosphorylation at ε-S435 and ε-S525 is directly proportional to growth. Phosphorylation of ε-S435 is necessary for effect of eIF2alpha-Ser51 phosphorylation on protein synthesis while phosphorylation of ε-S528 seems to be a target of various mechanisms. This study also suggests that eIF2Bε may be a key player of the cell cycle progression and phosphorylation changes can serve as marker for the regulation of eIF2B activity. The kinases responsible for phosphorylation at these sites are not yet known in yeast. Further investigation is required to find the functional significance of alterations in phosphorylation pattern to definitively establish eIF2Bε phosphorylation as a mechanism for regulating eIF2B activity in yeast. Models are presented to account for the results obtained that show how phosphorylation of eIF2Bε at these sites may contribute to the control of protein synthesis.

Type I PIP kinases (phosphatidylinositol 4-phosphate 5-kinases, PIP5Ks) catalyse the majority of cellular synthesis of PI(4,5)P2. To date, three mammalian isoforms (r1, r2, r3) have been found. PIP5KIr is subject to complex C-terminal splice variation, enhancing its transcriptional diversity through evolution and producing at least 5 known spliceoforms in the mammals. This study addresses several important questions. (1) Several remarkable differences have been discovered between the neuronal splice variant PIP5KIr_i3 and its close variant, Ir_i2, whose peptide lacks a 26-AA insert near its C-terminus. This study attempts to map these behavioural differences onto motifs within the peptide insert. Furthermore, a site of point mutation is identified near the activation loop, which amplifies the above differences. (2) This study documents properties of the more recently discovered PIP5KIr_i3, about which relatively little is known, for example, the regulation of its subcellular localisation, kinase activity and post-translational modifications. By site-directed mutagenesis and examining more closely several crucial motifs, insight is gained into the putative relationship between the enzyme’s phosphorylation state, cellular localisation, lipid kinase activity and autophosphorylation. (3) The discovery of a new PIP5KIr splice variant, Ir_v6, is described. First discovered in rodents, PIP5KIr_i6 encompasses the 26-AA insert of Ir_i3, but lacks the common C-terminus of Ir_i2 and Ir_i3 which contains peptide motifs that have several roles in vivo. A polyclonal antibody against the C-terminus of Ir_i6 was also developed. Preliminary characterisation of Ir_i6 demonstrates a similar subcellular localisation, but a wider expression profile than its close relative, Ir_i3, suggesting potentially differential functions across tissues and at various developmental stages. (4) The existence of Ir_v3 and Ir_v6 is also confirmed in humans. In light of recent findings of other novel human spliceoforms, this is shown to be a case of intra-exonic splicing producing “alternative 5’ splice site” exons in the human. Overall, this thesis should help to better understand the regulation and physiological roles of PIP5KIr and, specifically, its different splice variants.

Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2).

Correct regulation of transcription is essential for maintaining a healthy cellular state. During transcription RNA polymerase II (Pol II) proceeds in a regulated manner through several transitions to ensure appropriate control of synthesis and enable correct processing of the pre-RNA. Shortly after initiation Pol II is caused to pause by the binding of factors, DSIF and NELF. To enable transition of Pol II into the elongation phase CDK9/cyclin T phosphorylates the C-terminal domain (CTD) of Pol II, DSIF and NELF. This phosphorylation releases the paused state and provides an alternative set of post-transcriptional modifications on the CTD to generate a binding platform for elongation, histone modifying and termination factors. CDK9/cyclin T is itself regulated within multicomponent complexes. A small activated complex, containing Brd4, recruits CDK9/cyclin T to active sites of transcription, thereby promoting the elongation of transcription. The role of CDK9/cyclin T in the regulation of transcription has resulted in its validation as a drug target against several disease states including cancer, HIV and cardiac hypertrophy. In this thesis, I present the crystallographic structures of a series of 2-amino-4-heteroaryl-pyrimidine compounds and the roscovitine derivative, (S)-CR8, bound to CDK9/cyclin T and CDK2/cyclin A. In combination with thermal denaturation data and kinetic analysis, these structures have suggested chemical modifications that might be made to increase the CDK9 specificity of these compounds. I have also validated the use of a mutated form of cyclin T for use in the development of CDK9/cyclin T inhibitors. In addition, I present both structural and kinetic analysis of the Brd4-CDK9/cyclin T interaction. I show that C-terminal fragments of Brd4 enhance the in vitro kinase activity of CDK9/cyclin T against the Pol II CTD. Furthermore, I demonstrate that this enhancement may be inhibited by Plk1-mediated phosphorylation of Brd4. Finally, I show that Brd4 binds to a site that spans CDK9 and cyclin T and I propose detailed molecular models of the Brd4-cyclin T interaction.

Palmitoylation is a post-translational modification that has been implicated in the control of multiple proteins, including ion channels. S-Palmitoylation is a lipophilic modification that involves the attachment of palmitate through a thioester linkage to a cysteine residue in a target protein. By increasing the hydrophobicity of the target region, palmitoylation can promote membrane targeting. Here, palmitoylation is shown to play an important role in regulating large conductance calcium- and voltage- activated (BK) potassium channels. The STREX splice variant of the BK channel contains a 58 amino acid insert at the splice site C2 within the intracellular C-terminal RCK1-RCK2 linker that confers increased calcium sensitivity to the channel and determines PKA inhibition of channel activity. The cysteine rich STREX domain was predicted to be palmitoylated, and using an imaging assay STREX was shown to act as a membrane targeting domain through palmitoylation of a di-cysteine motif (C645:C645). A membrane potential assay and electrophysiological analysis demonstrates that palmitoylation at the C645:C646 site in STREX is important in mediating the increased calcium sensitive properties inherent to the STREX channel. Palmitoylation is also shown to modulate PKA channel inhibition. The stability of palmitoylation can often be reliant on the local environment within the protein. Generally in most proteins; lipidated regions, basic domains or transmembrane domains are found adjacent to a palmitoylation site. In STREX, a polybasic domain composed of 11 basic residues just upstream from the C645:C646 palmitoylation site, functions to control the palmitoylation status of the STREX insert. A site directed mutagenesis approach to disrupt the polybasic domain revealed an important role in controlling membrane targeting of the STREX C-terminus, mediating the increased calcium sensitivity inherent to STREX channels and controlling the palmitoylation status of the C645:C646 palmitoylation site using multiple techniques involving electrophysiology, fluorescent imaging and biochemical assays. Further to this, using imaging to examine the membrane association of fluorescently tagged C-terminal proteins, phosphorylation is shown to function as a physiological electrostatic switch to regulate the polybasic region in controlling palmitoylation of the STREX insert. Finally, an additional palmitoylation site that is constitutively expressed in all BK channels was identified to be located in the S0-S1 linker (C53:C54:C56). Mutation of the C53:C54:C56 palmitoylation site in the S0-S1 linker was shown to abolish all palmitoylation in BK channels that did not contain the STREX insert. Palmitoylation allows the S0-S1 linker to associate with the plasma membrane however the mutated de-palmitoylated channels did not affect channel conductance or the calcium/voltage sensitivity of the channel. Palmitoylation of the S0-S1 linker was shown to be a critical determinant of cell surface expression of BK channels, as steady state surface expression levels were reduced by ~55% in the C53:C54:C56 mutant. STREX channels that could not be palmitoylated in the S0-S1 linker also showed decreased surface expression even through STREX insert palmitoylation was unaffected. Palmitoylation is rapidly emerging as an important post-translational mechanism to control ion channel behaviour. This work reveals that palmitoylation of the BK channel can control channel function of the STREX splice variant channel and can regulate cell surface expression in all other channel variants. Palmitoylation appears to be functionally independent at these two distinct sites expressed within the same channel protein.

Deubiquitinating enzymes (DUBs) constitute a diverse protein family. The specificities and functions of the majority of DUBs are unknown, although their impact on many biological and pathological processes is widely appreciated. This dissertation entails a detailed characterization of otubain 1 (OTUB1), an ovarian tumor domain-containing DUB. The presented work describes OTUB1’s specificity, localization, protein interactions, importance in infection with Yersinia, and proposes a novel model of regulation of its enzymatic activity. I first discuss the structural and biochemical properties of OTUB1, demonstrating its selectivity towards ubiquitin and NEDD8. Moreover, I show that OTUB1 cleaves lys48- but not lys63-linked polyubiquitin, emphasizing its role in ubiquitin-mediated proteasomal degradation. Mass spectrometric identification of interaction partners and localization studies suggest possible involvement of OTUB1 in RNA processing and cell morphology. Furthermore, I demonstrate that invasion of the host cells by the enterobacteria Yersinia can be altered by changing OTUB1 expression. This effect is dependent on the catalytic activity of OTUB1 and its ability to stabilize RhoA-GTP prior to infection. YpkA and OTUB1 modulate RhoA-GTP stability in opposing ways, leading to cytoskeletal rearrangements that may be involved in bacterial invasion. Moreover, OTUB1 is post-translationally modified by phosphorylation that modulates its ability to stabilize RhoA-GTP and counteracts its effect on bacterial invasion. These findings provide a novel entry point for the manipulation of the host—pathogen interactions. Lastly, a kinase screen revealed that FER, an oncogenic kinase with a role in cell morphology, phosphorylates OTUB1, as demonstrated by overexpression, siRNA and in vitro studies. The phosphorylated site was mapped to tyr26 and the activity-based labeling revealed that this modification interferes with the deubiquitinating activity of OTUB1. In summary, the results presented in this thesis confirm that OTUB1 exerts properties of a “classical DUB” and uncover some of its physiological functions.

During stress conditions, glucocorticoids are secreted and exert most of their physiological responses by binding to and modulating the transcriptional activity of the glucocorticoid receptor (GR). Once activated, GR can regulate numerous cellular processes including inflammation, development, growth, metabolism and apoptosis. Although glucocorticoids have been used in the treatment of leukaemia for over 50 years, with the molecular mechanisms by which steroids exert their pro-apoptotic effect, the pathways responsible for the development of resistance to glucocorticoid treatment, as well as their role in the programmed cell death in other tissue types have not been precisely defined. Research has demonstrated that glucocorticoid-induced apoptosis requires a transcriptionally active form of GR and is executed by the induction of the intrinsic pathway of apoptosis. In addition, GR is regulated by diverse types of cytotoxic stress; including UV irradiation and hypoxia, which alter the receptor’s transcriptional activity through multiple mechanisms. These include post-translational modifications, subcellular localisation and interaction of the receptor with co-regulator proteins. The aims of this study are to identify novel members of the Bcl-2 family that are regulated at the transcriptional level by GR in both leukaemia and other tissue types where glucocorticoids promote cell survival. In addition, the molecular crosstalk between signalling pathways activated by cytotoxic stress conditions and the mechanisms by which they differentially modulate the apoptotic response will be investigated. Results obtained in this study have identified putative glucocorticoid response elements in the promoters of the BH3-only pro-apoptotic gene NOXA and the anti-apoptotic gene Mcl-1 and confirmed that both NOXA and Mcl-1 are direct GR transcriptional targets. The glucocorticoid-mediated expression of NOXA and Mcl-1 alters their protein-protein interaction pattern, leading to the subsequent destabilisation of Mcl-1 in cell lines that undergo glucocorticoid-induced apoptosis. Investigation into the effects that other cytotoxic stress pathways have on GR function have revealed that serine 226 phosphorylation of GR by JNK occurs in a rapid and transient manner. Phosphorylation has inhibitory effects on the transcription of GR targets in a gene-specific manner, including the differential regulation of NOXA gene expression. During hypoxia, glucocorticoids differentially regulate the GR and HIF-1 target genes, NOXA and Mcl-1, altering the apoptotic response. This study has provided additional insight into the molecular mechanisms that govern glucocorticoid-induced programmed cell death and revealed mechanisms by which glucocorticoids and cytotoxic stress pathways crosstalk, regulating apoptosis.

The application of the proteomic approach has facilitated efforts directed toward the mapping of the entire Arabidopsis cell wall proteome. Proteins were sequentially extracted from purified cell walls using 0.2 M CaC1(_2) followed by a urea buffer. The extracts were resolved via large format two dimensional polyacrylamide gel electrophoresis (2-D PAGE) and were visualised via Coomassie brilliant blue staining. The aim was to identify and characterise as many cell wall proteins as possible, with the hope of identifying novel cell wall proteins. Out of 325 spots visualised on the 2-D polyacrylamide gel, 144 gave a positive protein identification representing 104 different proteins. The identified proteins were divided into 3 categories. The first category included proteins that have been previously identified as plant cell wall proteins. The second category was designated to include novel cell wall proteins (hypothetical proteins) and the third category was made up of proteins, which had recognised functions, but had never hitherto been known to be secreted to the extracellular matrix. Among the identified novel cell wall proteins there were several that shared high homology with protein kinases. These proteins possessed all the characteristics of secreted polypeptides, such as the cleavable N-terminal signal peptide, and were found to lack both the transmembrane domain and the endoplasmic reticulum retention tetrapeptides (HDEL and KDEL). These observations suggested that, as in animal cells, plant cells had extracellular protein kinase activity (phosphorylation). This was supported by the recent discovery that plant cells secrete ATP to the extracellular matrix (Thomas et al., 2000). Verification of the occurrence of extracellular protein kinase activity was further strengthened by the identification of phosphorylated bona fide cell wall proteins and stress responses caused by the depletion extracellular ATP.

This thesis describes the analysis of the psbH gene structure and function in the unicellular green alga Chlamydomonas reinhardtii. The psbH gene encodes a small photosynthetic protein which co-purifies with Photosystem II (PS II). It is present in both cyanobacterial and eukaryotic PSII and is phosphorylated in the latter. The function of the phosphoprotein is still a matter of conjecture. Prior to the initiation of this thesis the gene had not been mapped or isolated in Chlamydomonas reinhardtii. This thesis describes the localisation of the psbH gene and its position relative to other genes already mapped. The complete sequence of the psbH gene was determined, the protein has a predicted molecular mass of 9.3 kDa. Northern blot analysis of the gene expression in wild type and nuclear mutants of C.reinhardtii reveal that the gene is co-transcribed with psbB located 3.2kB downstream from psbB and that a nuclear factor is required for stable expression of both genes. The precise location of the psbH transcripts revealed in northern analysis was mapped by primer extension. A series of psbH mutants in C.reinhardtii were obtained using a reverse genetics approach. The psbH gene was cloned into a suitable vector and an aadA cassette conferring resistance to the antibiotics spectinomycin and streptomycin was placed either within the psbH gene hence entirely knocking out gene function or just downstream of the gene. In the latter of the constructs, a site directed change was made within the gene resulting in a threonine to alanine substitution at the predicted phosphorylation site. Wild type C.reinhardtii cells were then transformed with this construct using the biolistic technique. Transformants were selected for by growth in media containing spectinomycin. Initial analysis of the mutants using Electron Paramagnetic Resonance studies, fluorescence analysis, and oxygen evolution analysis reveals that complete loss of the phosphoprotein results in loss of photosynthetic function for the alga but loss of the phosphorylation site alone does not lead to loss of photosynthetic function.