SRMS (Src-Related tyrosine kinases lacking C-terminal Regulatory tyrosine and N terminal Myristoylation Sites) belongs to a family of non-receptor tyrosine kinases, which also includes breast tumor kinase (BRK). SRMS was first identified in 1994 in a screen for the genes that regulate the growth and differentiation of neuroepithelial cells. This 54 kDa protein spanning 488 amino acids, consists of the prototypical Src homology 3 (SH3), Src homology 2 (SH2) and a tyrosine kinase domain. While BRK has been documented for its expression in over 60 % of breast carcinomas, information on SRMS on similar grounds remains absent from the literature. Furthermore, unlike BRK, knowledge of how SRMS regulates its enzymatic activity as well as the identification of its substrates remains unknown. The work in this thesis demonstrates that SRMS is potentially expressed in the majority of breast carcinomas. To understand the biochemical and cellular functions of SRMS, a series of mutants comprising point mutations as well as the deletion of the N-terminal region and the functional, SH3 and SH2 domains, were generated and assessed for enzymatic activity in cells. This study demonstrates for the first time that the wild type protein is apparently constitutively active and that its N-terminal region regulates its enzymatic activity. As well, three critical amino acid residues in the protein namely, lysine 258 (ATP binding site), tyrosine 380 (auto-phosphorylation site) and tryptophan 223 (intramolecular interaction) have been characterized. All three residues have been determined to be essential for the enzymatic activity of SRMS. Finally, the adapter protein Dok1 has been characterized as a novel substrate of SRMS. The results from the present study underscore the potential significance of the catalytically active non-receptor tyrosine kinase, SRMS that should serve as a foundation upon which further research may ensue in the context of breast tumorigenesis.

Within the cell nucleus, there are regions of highly condensed, transcriptionally silent chromatin called heterochromatin. Heterochromatin plays an important role in both chromosomal stability and gene regulation within the cell. Heterochromatin assembly is mediated by Heterochromatin Protein 1 (HP1) binding to epigenetically marked histone tails, most notably methylated H3K9. HP1 is post-translationally phosphorylated at serine and threonine residues, and this phosphorylation has been shown to increase HP1’s binding affinity for methylated H3K9 and heterochromatin formation. To study the effect of phosphorylation on heterochromatin assembly and HP1 localization within the nucleus, the unicellular protozoan Tetrahymena thermophila was used. Tetrahymena is an ideal model for this work because cells have a dynamic chromatin environment. Tetrahymena have an HP1-like protein, tHP1, which localizes to transcriptionally silent chromatin bodies within the otherwise transcriptionally active macronucleus. tHP1 is known to be phosphorylated at threonine-64 (site one) and at either serine-102 or threonine-103 (site two). Previous work shows that when phosphorylation at both sites is prevented, tHP1 exhibits decreased localization to chromatin bodies. In order to determine which site of phosphorylation accounts for tHP1’s localization to regions of heterochromatin, mutant proteins that allow phosphorylation at only one of the two sites were generated. The efforts to engineer a mutant protein that cannot be phosphorylated at site two and to visualize the protein’s localization throughout cell development are discussed. When phosphorylation is prevented at site two, tHP1 localization to regions of heterochromatin remains intact. These results suggest that phosphorylation at site one, not site two, may be responsible for tHP1 localization to macronuclear chromatin bodies. A mechanism by which site one phosphorylation influences tHP1 targeting to regions of heterochromatin is proposed. Furthermore, bioinformatics techniques are employed to identify other tHP1-like proteins within Tetrahymena. Characterization of these proteins will likely contribute to a more complete model of how heterochromatin is assembled in Tetrahymena.

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, which catalyses the second committed step in cholesterol biosynthesis. The mechanism by which SPF stimulates this enzyme is not understood and the goal of these studies was to see if SPF affected cholesterol synthesis in cultured cells. Rat supernatant protein factor-like protein (SPF2) shares 77% sequence identity with human SPF. In my studies SPF2 also stimulated squalene monooxygenase in vitro and incubation of SPF2 with protein kinase A (PKA) and C increased its activity by about 2-fold, as shown earlier with SPF. GTP and GDP prevented the stimulation of squalene monooxygenase by SPF2, suggesting that binding of these nucleotides inhibits SPF2. This inhibition could be prevented by the addition of -tocopherol, although -tocopherol alone had no effect on SPF2 activity in vitro. Expression of human SPF in hepatoma cells, which lack expression of endogenous SPF, increased cholesterol synthesis by 2-fold and addition of dibuytrylcAMP, a PKA activator, to these cells yielded an additional 62% increase whereas addition of a PKA inhibitor completely blocked the ability of SPF to stimulate cholesterol synthesis. To further confirm a role for phosphorylation in the regulation of SPF, substitution of alanine for serine-289 (a putative PKA recognition site) reduced the PKA-mediated activation of SPF in vitro by 50%, as measured with microsomal squalene monooxygenase and completely blocked the ability of SPF to stimulate cholesterol synthesis in hepatoma cells. In further structure-function studies, deletion of the carboxy-terminal Golgi-dynamics domain greatly increased the ability of SPF to stimulate squalene monooxygenase in microsomes, but, paradoxically prevented SPF from stimulating cholesterol synthesis in cell culture. Addition of brefeldin A, which disrupts Golgi formation, also abolished the ability of SPF to stimulate cholesterol synthesis, supporting a role for the Golgi in SPF function. Since squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway was investigated. The substitution of 14Cmevalonate for 14C-acetate completely blocked an SPF-induced 1.5-fold increase in squalene synthesis, suggesting that SPF stimulated mevalonate synthesis at HMGCoA reductase. 2,3-Oxidosqualene synthesis from 14C-mevalonate remained elevated (1.3-fold) with mevalonate demonstrating that SPF also stimulated squalene monooxygenase in hepatoma cells. SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels in cells, indicating that SPF directly activated these enzymes. Indeed, addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and, coupled with the ability of PKA-mediated phosphorylation to regulate SPF activity, suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.

The class II Transactivator (CIITA) is the master regulator for Major Histocompatibility Class II (MHC II) molecules. CIITA is dynamically regulated by a series of Posttranslational Modifications (PTMs). CIITA is responsible for initiating transcription of MHC II genes, thus allowing peptides derived from extracellular antigens to be presented to CD4+ T cells. CIITA’s PTMs are necessary for regulation of CIITA’s location, activity, and stability. Our work identifies the kinase complex ERK1/2 as being responsible for phosphorylating the previously identified regulatory site, serine (S) 280 on CIITA. Phosphorylation by ERK1/2 of CIITA S280 leads to increased levels of CIITA mono-ubiquitination and overall increases in MHC II activity. We further identify a novel ubiquitin modification on CIITA, lysine (K) 63 linked ubiquitination poly ubiquitination. Our data shows novel crosstalk between K63 ubiquitination and ERK1/2 phosphorylation. K63 ubiquitinated CIITA is concentrated to the cytoplasm, and upon phosphorylation by ERK1/2, CIITA translocates to the nucleus, thus demonstrating that CIITA’s location and activity is regulated through PTM crosstalk. While ubiquitination has been shown to be a critical PTM in the regulation of CIITA, the enzyme(s) mediating this important modification remained to be elucidated. Previous reports implicating the histone acetyltransferase (HAT), pCAF as an ubiquitin E3 ligase were intriguing, as pCAF is also known to participate in the acetylation of both histones at the MHC II promoter and in acetylation of CIITA. We now identify novel roles for pCAF in the regulation of CIITA. We show pCAF acts as an E3 ligase, mediating mono, K63, and K48 linked ubiquitination of CIITA. We therefore demonstrate an additional substrate for the “dual acting” enzyme, pCAF. In sum, our observations identify enzymes involved in both the phosphorylation and ubiquitination of key residues of CIITA, which ultimately regulate CIITA activity. Together our observations contribute to knowledge of CIITA’s growing network of PTMs and their role in regulating the adaptive immune response, and will allow for development of novel therapies to target dysregulated CIITA activity during adaptive immune responses.

Immunosuppressants that target the serine/threonine phosphatase calcineurin are commonly administered following organ transplantation. Their chronic use is associated with reduced insulin secretion and new onset diabetes in a subset of patients, suggestive of pancreatic β cell dysfunction. Calcineurin plays a critical role in the activation of CREB, a key transcription factor required for β cell function and survival. CREB activity in the islet is activated by glucose and cAMP, in large part due to activation of Crtc2, a critical coactivator for CREB. Previous studies have demonstrated that Crtc2 activation is dependent on dephosphorylation regulated by calcineurin. In this study, we sought to evaluate the impact of calcineurin-inhibiting immunosuppressants on Crtc2-CREB activation in the primary β cell. In addition, we further characterized the role and regulation of Crtc2 in the β cell. We demonstrate that Crtc2 is required for glucose dependent up-regulation of CREB target genes. The phosphatase calcineurin and kinase regulation by LKB1 contribute to the phosphorylation status of Crtc2 in the β cell. CsA and FK506 block glucose-dependent dephosphorylation and nuclear translocation of Crtc2. Overexpression of a constitutively active mutant of Crtc2 that cannot be phosphorylated at Ser171 and Ser275 enables CREB activity under conditions of calcineurin inhibition. Furthermore, β cells lacking Crtc2 display impaired glucose-stimulated insulin secretion and cell survival. Together, these results demonstrate that phosphorylation of Crtc2 plays a critical role in regulating CREB activity and contributes to β cell dysfunction and death caused by chronic immunosuppression.

The enzyme carbonic anhydrase (CA) contributes to multiple physiological processes by catalysing the reversible hydration of carbon dioxide. However, regulation of CA activity in response to homeostatic challenges remains poorly understood. The objectives of this thesis were to investigate whether CA is transcriptionally regulated by cortisol in fish and whether post-translational modification (PTM) of CA occurs in fish. The results of an in vivo reporter assay used to investigate potential transcriptional regulation of zebrafish, Danio rerio, cytoplasmic CA (CAc) were inconsistent, and it remains unclear whether zebrafish CAc is regulated transcriptionally by cortisol. Phosphorylation of rainbow trout, Oncorhynchus mykiss, CAc was predicted from in silico analysis of the putative amino acid sequence and confirmed by Western analysis of phosphoprotein levels following in vitro incubation of CA, purified from trout gill, under conditions designed to potentiate endogenous kinases. Again using in vitro incubations designed to potentiate endogenous kinases and phosphatases, changes to the phosphorylation state of CAc were found to modulate its enzymatic properties. These findings suggest that CA activity may be regulated by signalling pathways that activate cellular protein kinases, and future work should focus on identifying these pathways.

Normal cellular behaviour in multicellular organisms is achieved by tight control of signaling pathway networks. The mitogen-activated protein kinase (MAPK) signaling cascade is one of these signaling networks, that when deregulated can lead to cellular transformation. Activation of the RAS-RAF-MEK-MAPK (ERK) signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Subsequently, MSKs go on to phosphorylate histone H3 at Ser10 and Ser28.Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Hras-transformed cells (Ciras-3) relative to these activities in the parental 10T1⁄2 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T1/2 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the RAS-MAPK signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in oncogene-transformed cells. Furthermore, upon activation of the ERK and p38 MAPK pathways, the MSK1/2- mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2, Ciras-3 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 (the ATPase subunit of the SWI/SNF remodeler) is recruited to the promoter of target genes by transcription factors such as ELK-1 or NFκB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of immediate-early genes enabling the binding of transcription factors like JUN and the onset of transcription. Since RAS-MAPK activated MSKs mediate H3 phosphorylation that is required for expression of various immediate-early gene products involved in cellular transformation, inhibition of MSK activity may be a therapeutic target that could be exploited in cancers with upregulated RAS-MAPK signaling.

The RAS-mitogen-activated protein kinase (MAPK) pathway is an essential signaling mechanism that regulates cellular processes and culminates in the activation of specific gene expression programs. Alterations in the RAS-MAPK signaling cascade can modify epigenetic programs and confer advantages in cell growth and transformation. In fact, deregulation of the cascade is a key event in tumour development with 30% of human cancers harbouring RAS mutations. In breast and pancreatic epithelial cancers, characterization of an aberrant RAS-MAPK pathway has focused on upstream mediators such as receptors and oncogenic RAS molecules but the impact of downstream targets remain poorly defined. Stimulation of the RAS-RAF-MEK-MAPK pathway leads to activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2) which are responsible for the phosphorylation of histone H3 on S10 and S28. We postulate that deregulation of the RAS-MAPK pathway produced by constitutive activation and/ or over-expression of upstream components or mitogen stimulation consequently leads to enhanced MSK1 activity and elevated histone H3 phosphorylation levels. We further hypothesize that MSK1-mediated H3 phosphorylation is critical for immediate early gene (IEG) expression, Ras-driven transformation and is associated with regulatory regions upon gene transcription. In mouse fibroblasts, we present evidence for the critical involvement of MSK1 and H3 phosphorylation as mediators that bridge the aberrant signals driven by the RAS-MAPK pathway with nucleosomal modifications, chromatin remodeling, IEG expression and malignant transformation. We then examined if activation of RAS-MAPK signaling in breast cancer cells elicits similar molecular events. We demonstrate that the RAS-MAPK pathway is induced and enhances the association of MSK1 and H3 phosphorylation on the IEG Trefoil Factor 1 resulting in transcriptional activation. We further observed that mutated K-RAS expression did not correlate with genomic instability or altered signaling in pancreatic cancer cell lines while overexpressed HER2 and EGFR breast cancer cell lines generally exhibit upregulated ERK1/2 and H3 phosphorylation levels. Taken together, our studies contribute to the further understanding of MSK-mediated transcriptional activation in response to RAS-MAPK signaling in oncogene-transformed and cancer cell lines. Inhibition of MSK activity may be an unexplored avenue for combination cancer therapy with abnormal RAS-MAPK signaling pathways.

Alternative pre-mRNA splicing is often controlled by cell signals (1-3). Membrane depolarization/calcium (Ca2+) signaling controls alternative splicing of a group of genes in neurons and endocrine cells (4-9), with important implications in memory formation or secretion of hormones and neurotransmitters (10-15). However, the underlying molecular basis remains largely unknown. In rat GH3 pituitary cells, BK potassium channels control cellular electrical firing, which is critical for the release of growth hormone and prolactin. Inclusion of the STREX exon of the Slo1 gene encoding the channel α subunit is repressed by the Ca2+/calmodulin-dependent kinase IV (CaMKIV) upon depolarization (4). We isolated CaMKIV-responsive RNA elements (CaRREs) from a library of 13-nucleotide random sequences through in vivo selection in HEK293T cells. Most elements are CA-rich or A-rich, with the heterogeneous nuclear ribonucleoprotein (hnRNP) L as a binding factor. This is consistent with the finding that CA-rich elements and hnRNP L are targeted by CaMKIV in the regulation of splicing (16). In further efforts to directly link the kinase with hnRNP L, we showed that hnRNP L is essential for the full repression of STREX by depolarization and that a highly conserved CaMKIV target serine (Ser513) of L is required. Ser513 phosphorylation enhanced L binding to the STREX CaRRE1, leading to reduced binding of the constitutive factor U2AF65 to the 3’ splice site of STREX. Mutation of Ser513 abolished both activities. Therefore, hnRNP L mediates the repression of STREX by depolarization through modulation of a key step in spliceosomal assembly. We further identified hnRNP L, L-like (LL) and PTB as repressors of STREX and other depolarization-regulated exons with differential effects. Moreover, a full response of STREX to depolarization is mediated by combinations of hnRNP L and LL or PTB. Another depolarization-responsive exon, the exon 18 of the neuregulin 1 gene, is also controlled in a similar way, with the hnRNP L Ser513 required as well. This work provides the first direct link between the Ca2+ signaling and a specific serine of a regulatory splicing factor. Elucidation of the underlying molecular mechanisms would likely help us understand the fine-tuning of hormone secretion and memory formation.

Cell polarity is fundamental to numerous cellular processes including migration, molecular transport, and cell division. The establishment and organization of polarity is crucial to the maintenance of cellular homeostasis in mammalian systems. Deregulation of cell polarity is observed in disease states, including cancer. Numb is an adaptor protein that functions in regulating endocytic trafficking events. Numb was originally identified in Drosophila as an asymmetrically localized cell fate determinant, and was subsequently found to be conserved in vertebrates. In mammalian polarized epithelial cells, Numb is distributed asymmetrically along the basolateral membrane domain. The work herein describes phosphorylation of Numb by the Par complex protein, atypical Protein Kinase C (aPKC), as a means of regulating membrane localization and asymmetric distribution of Numb. A mutant of Numb that cannot be phosphorylated by aPKC accumulates on the plasma membrane and localizes to both apical and basolateral membranes. In aPKC-depleted cells, endogenous Numb is unable to achieve polarized distribution and localizes around the entire cell cortex. We demonstrate that this mechanism is conserved in Drosophila as mutation of the corresponding phosphorylation sites disrupts Numb asymmetric localization in dividing sensory organ precursor cells. In polarized epithelial cells, one function of Numb is to promote epithelial morphology when cells are challenged with external stimuli that disrupt cell-cell adhesion. For example, depletion of Numb results in enhanced sensitivity of cells to lose cell-cell contacts when treated with calcium chelating agents. Loss of Numb potentiates hepatocyte growth factor (HGF)-induced lamellipodia formation and cell dispersal – early steps in epithelial-mesenchymal transition (EMT). In Numb-depleted cells, Rac1-GTP loading is enhanced, which corresponds with increased rate in loss of cell-cell adhesion and increased lamellipodia formation, following depletion of extracellular calcium and HGF stimulation, respectively. Together, this work identifies a mechanism that regulates polarized distribution of Numb and provides insight into its function in polarized epithelial cells.