The establishment of correct neural circuitry in the nervous system requires the interplay, integration, and coordination of a diverse set of cells and signals during development and in the adult. Two important events are the regulated initiation and growth of dendrites that receive and process synaptic information, and the establishment and maintenance of appropriate neural connectivity. The goals of this study are to identify the molecular mechanisms underlying dendrite growth and initiation, and to understand how neural connectivity is maintained in the adult nervous system. I first identified a novel intracellular signal transduction pathway involving two kinases important in regulating dendrite development. I showed that the ILK-GSK3beta pathway is required for dendrite growth and initiation in both peripheral and central nervous system neurons. I then asked how neural connectivity is maintained in the adult nervous system by examining the role of myelin in the intact nervous system. My results indicate that when myelin contacts aberrantly growing axons, it activates on those axons the p75 neurotrophin receptor (p75NTR), which in turn causes the local degeneration of those axons. I further identified the signal transduction pathway required for axon degeneration consisting of p75NTR and intracellular signaling proteins activated by this receptor, Rho-GDI, Rho, and caspase 6. This data establishes p75NTR as an important regulator of neural connectivity and identifies for the first time a degeneration-inducing signal transduction pathway activated by myelin. It also provides an explanation for why myelin inhibits regeneration of injured central nervous system axons. Taken together, I identified a new signaling pathway important for regulating dendrite initiation and growth, and a novel role for myelin in maintaining neural connectivity. Both of these findings contribute to our knowledge of how such connectivity is established during development and maintained in the adult nervous system.

The shikimate pathway links carbohydrate metabolism to the biosynthesis of the aromatic amino acids and an enormous variety of aromatic compounds with essential functions in all kingdoms of life. Aromatic compounds derived from the plant shikimate pathway have substantial biotechnological value and many are essential to the diet of metazoans whose genomes do not encode shikimate pathway enzymes. Despite its importance to the physiology of plants and human health the regulatory mechanisms of the plant shikimate pathway are not well understood. Shikimate kinase (SK) genes encode an intermediate step in the shikimate pathway and were previously implicated in regulation of the plant shikimate pathway. The distribution of SK genes in higher plants was resolved using phylogenetic and biochemical methods. The two SK isoforms of Arabidopsis thaliana, AtSK1 and AtSK2, were functionally characterized. AtSK1 expression is induced by heat stress and the recombinant enzyme was shown to form a homodimer which is important for maintaining the stability and activity of the enzyme at elevated temperatures. The crystal structure of AtSK2, the first reported plant SK structure, identified structural features unique to plant SKs which may perform important regulatory functions. The resolution of bona fide SKs in higher plants led to the discovery of two novel neofunctionalized homologs - Shikimate Kinase-Like 1 (SKL1) and SKL2. These novel genes evolved from SK gene duplicates over 400 million years ago and are found in all major extant angiosperm lineages, suggesting they were important in the development of biological properties required by land plants. The description of albino and variegated skl1 mutants in Arabidopsis thaliana implicate the SKL1 gene product as an important regulator of chloroplast biogenesis. Functional assays were attempted to determine the biochemical function of SKL1 and recombinant constructs of the Arabidopsis thaliana SKL1 protein were crystallized towards structure determination. The results of this thesis further our understanding of the organization and regulation of the plant shikimate pathway. Furthermore, the discovery of SKL1 may yield important insights into chloroplast biogenesis and function. The evolution of the plant SK superfamily highlights the utility of SKs as scaffolds for functional innovation.

The ubiquitination pathway regulates virtually all cellular processes such as cell cycle control and immune surveillance in eukaryotes, and is thus highly regulated through a variety of means. For instance, the Cullin-RING ubiquitin E3 ligases are regulated by neddylation through the action of a newly identified protein Dcn1. In chapter two, I describe an X-ray crystal structure of yeast Dcn1, encompassing an N-terminal ubiquitin association (UBA) domain and a C-terminal domain of unique architecture, which I termed the PONY (POtentiating NeddYlation) domain. I describe the identification of the reciprocal, conserved binding surfaces on both Dcn1 and yeast cullin Cdc53. In collaboration with Dr. Matthias Peter’s group (ETH Zurich), we show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Together, our data identify Dcn1 as the long sought-after Nedd8 E3 ligase for cullin neddylation. As a modulator of immune surveillance and inflammatory responses, the ubiquitin system serves as an attractive target for subversion by pathogens. In chapter three, I present a structural and functional analysis of a newly identified bacterial ubiquitin E3 ligase IpaH, present in various pathogenic and commensal bacteria. I demonstrate that the leucine-rich repeat (LRR) substrate recognition domains of different IpaH enzymes auto-inhibit the enzymatic activity of the adjacent catalytic domain by two distinct but conserved structural mechanisms. Auto-inhibition is required for the biological activity of two IpaH enzymes in a yeast model system. Retro-engineering of auto-inhibition into a constitutively active IpaH enzyme from Yersinia demonstrates that most of the infrastructure required to support auto-inhibition is evolutionarily conserved. In brief, my research provides insights into the mechanism of action of two newly identified protein ligases in the ubiquitination pathway, namely the Nedd8 E3 ligase Dcn1 and bacterial ubiquitin E3 ligase IpaH.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

Cytokinesis, the final step of cell division, is characterized by formation of a cleavage furrow that ingresses to separate the cell into two daughter cells. This process requires rearrangement of the cytoskeleton and addition of membrane to the growing furrow. The phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been implicated in regulating both actin dynamics and membrane trafficking and, thus, is uniquely poised to coordinate these different processes during cytokinesis. In this study, I show that PtdIns(4,5)P2 is involved in another aspect of cytokinesis: regulation of actomyosin contractility. Perturbing PtdIns(4,5)P2 levels in Drosophila spermatocytes caused constriction to fail and cleavage furrows to regress. Moreover, PtdIns(4,5)P2 hydrolysis is implicated in this process: inhibiting PLC or IP3R or chelating Ca2+ also caused defects in furrow ingression. In addition, I show that PLC and MLCK activities are required for myosin light chain phosphorylation and for proper myosin and actin localization to the cleavage furrow. Thus, I propose a model in which PtdIns(4,5)P2 hydrolysis-dependent Ca2+ release activates MLCK via Ca2+/calmodulin to maintain myosin filaments in the contractile ring and regulate cleavage furrow ingression. Furthermore, I show that PtdIns(4,5)P2 has a role in maintaining contractile ring components in the cleavage furrow that does not depend on PtdIns(4,5)P2 hydrolysis. I conclude that PtdIns(4,5)P2 regulates myosin contractility through a PLC-dependent pathway leading to myosin phosphorylation and is also involved in localizing contractile ring components to the furrow. Thus, PtdIns(4,5)P2 may coordinate signals leading to cytoskeleton rearrangement and actomyosin contractility during cytokinesis.

The GATA transcription factors consist of six family members that bind the consensus DNA binding element W-GATA-R, and are poorly characterized in the central nervous system (CNS). In this thesis we identify GATA4 to be expressed in the neurons and glia of normal murine and human embryonic and adult CNS with significant loss in Glioblastoma Multiforme (GBM). GBM is the most common and lethal primary brain tumour and exhibits multiple molecular aberrations. Here we report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumour suppressor gene. Although GATA4 was expressed in normal brain, loss of GATA4 was observed in GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor like phenotype, formation of neurospheres and the ability to differentiate into astrocytes, neurons and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures and brain tumour initiating cells suppressed tumour growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT, the DNA repair enzyme often implicated in temozolomide resistance. Instead GATA4 reduced expression of APNG, a DNA repair enzyme poorly characterized in GBM mediated temozolomide resistance. Identification and validation of GATA4 as a tumour suppressor gene and its downstream targets in GBM may yield promising novel therapeutic strategies.

Much of the work on how the cell cycle is regulated has focused on Cyclin-Dependent Kinase (CDK)-mediated regulation of factors that control the coordinate expression of genes required for entry into the cell cycle. In Saccharomyces cerevisiae, SBF and MBF are related transcription factors that co-ordinately activate a large group of genes at the G1/S transition, and their activation depends on the Cln3-Cdk1 form of the cyclin-dependent kinase. However, cells are viable in the absence of Cln3, or SBF and MBF, indicating that other regulatory pathways must exist that activate the budding yeast cell cycle. The known CDK-independent pathways are made up of various phosphatases and plasma membrane transporters that control ion homeostasis in early G1 phase, a time when cells assess environmental growth conditions in order to commit to cell cycle entry. The enigmatic Bck2 protein is thought to act within these CDK-independent pathways, but the means by which it activates G1/S-regulated genes is not known. Bck2 contains little sequence homology to any known protein. In order to understand how CDK-independent pathways operate, I have studied the Bck2 protein using multiple approaches. In one approach, I have screened for novel SBF/MBF-binding proteins in order to determine if other non-CDK proteins, such as Bck2, might activate SBF and MBF. I have also investigated which region of Bck2 is required for its activity in order to determine if Bck2’s transcriptional activation region is essential. Using one of the iii truncation derivatives from this analysis, I have screened for proteins that interact with Bck2. One of these novel proteins is Mcm1, a global transcriptional activator of genes involved in cell cycle progression, mating gene transcription and metabolism. My studies suggest that Bck2 regulates the activity of Mcm1 in early G1 phase to activate the expression of SWI4, CLN3, and others. My evidence suggests that Bck2 competes for binding to a specific pocket on Mcm1 that is also bound by an Mcm1 repressor called Yox1. My findings suggest that CDK-independent pathways function through Bck2, in order to induce the initial suite of genes required for entry into the cell cycle.

The Hedgehog (Hh) signaling pathway has received attention regarding its important role in embryonic development, however the mechanism by which pathway regulators, such as Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh signaling transduction is not entirely understood. The work presented here examines the roles of Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and primary cilium are dispensable in this process. Although primary cilium is required for Hh ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional activity in a cilia-independent manner. The results of this work provide insight into how Hh signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte development.

Protein phosphorylation is a prevalent reversible post-translational modification that influences protein functions. The advent of phospho-proteomic technologies now enables proteome-wide quantitative detection of residues phosphorylated under different physiological conditions. The functional consequences of the majority of these phosphorylation events are unknown. This calls for endeavors to characterize their molecular functions and cellular effects. This can be facilitated by systematic approaches to categorize phosphorylation events, interpret their importance and infer their functions. I carried out comparative, evolutionary and integrative analyses on in vivo phosphorylation events to address these challenges. First, I performed cross-species comparative phospho-proteomic analysis to identify evolutionarily conserved phosphorylation events in human. A sequence alignment approach was used to identify phosphorylation events conserved at similar sequence positions across orthologous proteins and a network alignment approach was applied to identify potential evolutionarily conserved kinase-substrate interactions. Conserved human phosphoproteins identified are found enriched for proteins encoded by known cancer- and disease-associated genes. Next, I developed a new approach to analyze the sequence conservation of known phosphorylated residues on human, mouse and yeast proteins that factored in the background mutational rates of protein and phosphorylatable residue. Furthermore, sites were analyzed according to (i) characterized functions, (ii) prevalence, (iii) stoichiometry, their occurrence in (iv) structurally disordered/ordered protein regions, in (v) proteins of various abundance and in (vi) proteins with different protein interaction propensity to identify the factors influencing sequence conservation of phosphorylated residues. Importantly, my analysis suggests that false positives and randomly phosphorylated residues are present in existing phosphorylation datasets and they are more common on high abundance proteins. Lastly, I characterized the theoretical maximum phosphorylation capacity in terms of phosphorylatable residues and discovered that genomic tyrosine frequency correlates negatively and significantly with tyrosine kinase frequency and cell type in metazoan. This observation suggests that fidelity of phosphotyrosine signaling occurred partially through global tyrosine depletion.

The innate immune response is a primary transcriptional defence network activated by interferons (IFNs) α/ β in response to viral infection. A cell must have the capability to detect the virus, activate signalling cascades, and engage transcriptional anti-viral networks. IFNs trigger the Signal Transducer and Activator of Transcription (STAT) family, which in turn induce anti-viral gene expression. Recruitment of STATs to IFN stimulated gene (ISG) promoters and the ensuing gene induction requires Brahma Related Gene 1 (BRG1), the catalytic component of the SWI/ SNF chromatin remodelling (or BAF) complex. Cell lines with high BRG1 expression are hyper-responsive to IFN induced transcription, conversely BRG1 low cells exhibit impaired induction. However, BRG1 high cells that are resistant to Encephalomyocarditis virus infection did not require signalling through the IFN receptor complex for anti-viral immunity. This suggested 2F-BRG1 cells must rely on BRG1 dependent non-ISGs or an as yet uncharacterized subset of basally expressed BRG1-dependent ISGs that do not require IFN enhanced expression for anti-viral activity. Utilizing genome wide microarrays we identified five genes with potent anti-viral activity. These genes may restrict viral infection through alterations in integrin signalling, endosomal trafficking, and activation of host transcriptional responses. We also investigated the role of Special AT-Rich Binding Protein (SATB1) in regulation of IFN responsive genes. The loss of this chromatin binding protein is associated with transcriptional changes in the MHC locus that mimic IFNγ induced expression. Through microarray analysis we discovered a remarkable 47% of IFNα regulated genes were co-regulated by SATB1; 42% of IFNα induced genes were induced by SATB1 knock down, while 63% of IFNα repressed genes were SATB1 dependent. Functionally, knock down of SATB1 protected cells from EMCV induced cell death at low multiplicity of infection (MOI), and increased the cytoprotective effect of IFNα against EMCV at higher MOIs. Analysis of IFNα, SATB1 and BRG1 regulated genes revealed a subset of core genes regulated by all three factors that may be critical to robust anti-viral immunity. The potent immunosuppressive properties of SATB1 suggest this protein may be involved in complex immunopathologies. The immuno-modulatory properties of SATB1 and BRG1 established in this thesis provide substantive evidence for the development of pharmaceutical therapies targeting these proteins.