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ABSTRACT The voltage-gated potassium channel Kv1.2 is a key determinant of cellular excitability in the nervous and cardiovascular systems. In the brain, Kv1.2 is strongly expressed in neurons of the hippocampus, a structure essential for learning and memory, and the cerebellum, a structure essential for motor control and cognition. In the vasculature, Kv1.2 is expressed in smooth muscle cells where it contributes to the regulation of blood flow. Dynamic regulation of Kv1.2 is fundamental to its role in these tissues. Disruption of this regulation can manifest in a range of pathological conditions, including seizure, hypertension and neuropathic pain. Thus, elucidating the mechanisms by which Kv1.2 is regulated addresses fundamental aspects of human physiology and disease. Kv1.2 was the first voltage gated ion channel found to be regulated by tyrosine phosphorylation. The ionic current of Kv1.2 is suppressed following tyrosine phosphorylation by a process involving channel endocytosis. Movement of channel away from the plasma membrane involves many proteins associated with the cytoskeleton, including dynamin, cortactin and RhoA. Because trafficking of Kv1.2 away from the cell surface has emerged as the primary mechanism for its negative regulation, we hypothesized that trafficking of the channel to the cell surface could be a mechanism for positive regulation of the Kv1.2 ionic current. Activation of the cAMP/PKA pathway enhances the ionic current of Kv1.2. We hypothesized that a mechanism for this positive regulation is an increase in the amount of channel protein present at the cell surface. Our data show that cAMP can regulate Kv1.2 surface levels by two opposing trafficking pathways, one PKA-dependent and one PKA-independent. Channel homeostasis is preserved by the dynamic balance between these two pathways. Accordingly, any change in the levels of cAMP causes a net increase in the amount of Kv1.2 present at the cell surface. Specific C-terminal phosphorylation sites of Kv1.2 were identified and shown to have a role in maintaining basal surface channel levels. These findings demonstrate channel trafficking as a mechanism for the positive regulation of the Kv1.2 ionic current. In addition to Kv1.2 trafficking at the plasma membrane, movement of the channel from the biosynthetic pathway to the cell surface is another checkpoint for its regulation. Here we show that the protein arginine methyltransferase 8 (PRMT8) is able to promote the ER exit of Kv1.2, resulting in an increase in Kv1.2 surface expression. PRMT8 not only promoted surface expression of the high mannose glycosylated form of Kv1.2, characteristic of immature, ER-localized channels, but also enhanced Kv1.2 total protein levels, most likely by decreasing the amount of channel protein available for ER-associated degradation (ERAD). These findings highlight biosynthetic trafficking of Kv1.2 as a crucial part of its regulation and identify a novel role for PRMT8, as a regulator of biosynthetic protein trafficking.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. October 2014. / The c-Jun N-terminal kinases (JNKs) are mitogen-activated protein kinases (MAPKs) that are activated by the dual phosphorylation of a canonical threonine and tyrosine residue. While it is well known that the activation of JNK mediates many important cellular processes such as differentiation, proliferation, and apoptosis, the mechanisms by which phosphorylation induces its activation are not known. An understanding of the structural and biophysical basis for the activation of JNK is highly desirable however, as dysregulation of the kinase has been implicated in numerous prominent diseases. Aiming first to improve upon the previously reported inadequacies in acquiring active JNK, this work describes a novel method for the purification of large yields of pure and phosphorylated JNK1β1, the most abundant JNK isoform. Using codon harmonization as a precautionary measure toward increasing the soluble overexpression of the kinase raised unique questions about the role of translation kinetics in both the heterologous and natural co-translational modification of kinases. After purifying the upstream activating kinases of JNK, phosphorylation of JNK1β1 was achieved by reconstituting the MEKK1 → MKK4 → JNK MAPK activation cascade in vitro. Activated JNK1β1 was thereafter able to phosphorylate its substrate, ATF2, with high catalytic efficiency. Characterising the nature of JNK1β1 modification by MKK4, mass spectrometry revealed that the latter kinase phosphorylates JNK1β1 not only at its activation residues (T183 and Y185), but also at a recognised yet uncharacterised phospho-site (S377) as well as two novel phospho-residues (T228 and S284) whose phosphorylation appear to have functional significance. Unfolding studies and amide hydrogen-deuterium exchange (HX) mass spectrometry (MS) were then used to investigate the changes to the stability and structure/conformational dynamics of JNK1β1 induced by phosphorylation and nucleotide substrate binding. Increased flexibility detected at the hinge between the N- and C-terminal domains upon phosphorylation suggested that activation may require interdomain closure. Patterns of solvent protection by the ATP analogue, AMP-PNP, reflected a novel mode of nucleotide binding to the C-terminal domain of a destabilised and open domain conformation of inactive JNK1β1. HX protection at both domains following AMP-PNP binding to active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, simultaneously stabilising the kinase. This reveals that phosphorylation activates JNK1β1 in part by enhancing the flexibility of the hinge to enable interdomain closure and the formation of a functional active site. This work thus offers novel insight into the unique molecular mechanisms by which JNK1β1 is regulated by nucleotide binding and phosphorylation by MKK4, and by the complex interplay that exists between them.

by Yin Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 146-164). / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.iv / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter 1.1 --- Drosophila as a model for studying development --- p.1 / Chapter 1.2 --- The formation of the body axes of Drosophila --- p.3 / Chapter 1.2.1 --- Oogenesis --- p.5 / Chapter 1.2.2 --- Embryogenesis --- p.15 / Chapter 1.2.3 --- Segmentation --- p.16 / Chapter 1.2.4 --- Life cycle --- p.20 / Chapter 1.3 --- Egg-polarity genes are essential for development --- p.22 / Chapter 1.4 --- Maternal gene bicoid is required for formation of anterior structures in the embryo --- p.24 / Chapter 1.4.1 --- Phenotypes of bicoid mutant --- p.24 / Chapter 1.4.2 --- Transplantation experiment --- p.26 / Chapter 1.4.3 --- Establishment of an anterior to posterior bicoid protein gradient --- p.26 / Chapter 1.4.4 --- Localization step of bicoid mRNA --- p.27 / Chapter 1.4.5 --- Formation of bicoid protein gradient --- p.28 / Chapter 1.4.6 --- The bicoid protein gradient regulates the downstream zygotic target genes in a concentration-dependent manner --- p.31 / Chapter 1.4.6.1 --- Bicoid protein acts as transcriptional regulators --- p.31 / Chapter 1.4.6.2 --- Bicoid protein acts as transcriptional activators --- p.31 / Chapter 1.4.6.3 --- Bicoid protein acts as translational repressor --- p.34 / Chapter 1.5 --- Components required for the localization of bicoid mRNA --- p.35 / Chapter 1.5.1 --- Cis-acting elements --- p.35 / Chapter 1.5.1.1 --- Bicoid mRNA localization element (BLE1) at 3、UTR directs localization of bicoid mRNA --- p.36 / Chapter 1.5.2 --- Trans-acting elements --- p.37 / Chapter 1.5.2.1 --- exuperantia --- p.40 / Chapter 1.5.2.2 --- swallow --- p.41 / Chapter 1.5.2.3 --- staufen --- p.42 / Chapter 1.5.2.4 --- cytoskeleton --- p.44 / Chapter 1.6 --- Aim of project --- p.48 / Chapter CHAPTER 2 --- Characterization of exuperantia protein --- p.50 / Chapter 2.1 --- Introduction --- p.50 / Chapter 2.1.1 --- Localization step of exuperantia protein in wild type --- p.50 / Chapter 2.1.2 --- Phenotype of exuperantia mutant --- p.51 / Chapter 2.1.3 --- exuperantia gene in both female and male flies --- p.52 / Chapter 2.2 --- Materials and Methods --- p.59 / Chapter 2.2.1 --- General characteristic of exuperantia protein --- p.59 / Chapter 2.2.1.1 --- Preparation of total ovary protein from the female and male flies --- p.59 / Chapter 2.2.1.2 --- Analysis of exuperantia protein by Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS - PAGE) and Western blotting --- p.60 / Chapter 2.2.2 --- Determination of the type of phosphorylation residues in exuperantia protein --- p.61 / Chapter 2.2.2.1 --- Preparation of immunoprecipitated exuperantia protein from ovary and testis --- p.61 / Chapter 2.2.2.2 --- Dephosphorylation of exuperantia protein --- p.62 / Chapter 2.2.3 --- Two-dimensional gel electrophoresis analysis of exuperantia protein --- p.63 / Chapter 2.3 --- Results --- p.65 / Chapter 2.3.1 --- General characteristic of exuperantia protein --- p.65 / Chapter 2.3.2 --- Determination of the type of phosphorylation residues in exuperantia protein --- p.67 / Chapter 2.3.3 --- Resolving the multiple phosphorylated isoforms of exuperantia protein by two-dimensional gel electrophoresis --- p.69 / Chapter 2.4 --- Discussion --- p.72 / Chapter CHAPTER 3 --- Determination of the type of kinase(s) phosphorylate exuperantia protein --- p.77 / Chapter 3.1 --- Introduction --- p.77 / Chapter 3.2 --- Materials and Methods --- p.83 / Chapter 3.2.1 --- Phosphorylation of recombinant exuperantia protein --- p.83 / Chapter 3.2.1.1 --- Immunoprecipitation of recombinant exuperantia protein and phosphorylation reaction --- p.83 / Chapter 3.2.1.2 --- Sequential phosphorylation reaction --- p.84 / Chapter 3.2.2 --- Inhibitory effect(s) of protein kinase inhibitors on phosphorylation of native exuperantia protein --- p.85 / Chapter 3.2.2.1 --- Incubation of ovaries with protein kinase inhibitors --- p.85 / Chapter 3.2.3 --- Phosphorylation of native exuperantia protein by endogenous protein kinase(s) --- p.86 / Chapter 3.2.3.1 --- Preparation of total tissue homogenate --- p.86 / Chapter 3.2.3.2 --- Endogenous kinase assay --- p.86 / Chapter 3.3 --- Results --- p.88 / Chapter 3.3.1 --- Phosphorylation of recombinant exuperantia protein by exogenous kinase(s) --- p.88 / Chapter 3.3.2 --- Inhibitory effect(s) of protein kinase inhibitors on phosphorylation of native exuperantia protein --- p.92 / Chapter 3.3.3 --- Phosphorylation of native exuperantia protein by endogenous protein kinase(s) --- p.94 / Chapter 3.3.3.1 --- Phosphorylation of native exuperantia protein by endogenous kinase(s) with addition of protein kinase activators --- p.94 / Chapter 3.3.3.2 --- Phosphorylation of native exuperantia protein by endogenous kinase(s) with addition of protein kinase inhibitor --- p.98 / Chapter 3.4 --- Discussion --- p.101 / Chapter CHAPTER 4 --- Spatial and temporal distribution of exuperantia protein in DCO83 and exuPJ egg chambers --- p.107 / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Initiation of establishment of the two body axes by one single signal --- p.107 / Chapter 4.1.2 --- Stage-specific phosphorylation of exuperantia protein --- p.111 / Chapter 4.2 --- Materials and Methods --- p.113 / Chapter 4.2.1 --- Immunohistochemical distribution of exuperantia protein --- p.113 / Chapter 4.2.2 --- Stage-specific phosphorylation of exuperantia protein --- p.115 / Chapter 4.3 --- Results --- p.116 / Chapter 4.3.1 --- Immunohistochemical distribution of exuperantia protein in DCOB3 mutant --- p.119 / Chapter 4.3.2 --- Immunohistochemical distribution of exuperantia protein in exuPJ mutant --- p.121 / Chapter 4.3.3 --- Stage-specific phosphorylation of exuperantia protein in DCOB3 mutant --- p.125 / Chapter 4.3.4 --- Stage-specific phosphorylation of exuperantia protein of exuPJ mutant --- p.127 / Chapter 4.4 --- Discussion --- p.128 / Appendix A --- p.135 / Appendix B --- p.143 / References --- p.146

Mediators released from the granules of antigen-activated mast cells contribute to allergies, inflammation and diseases such as asthma. One of the major models used to study mucosal mast cells is the RBL-2H3 mast cell line. While there has been considerable research on the initial signalling events following IgE receptor (Fc??RI) cross-linking, the movement of granules to sites of exocytosis is poorly understood. Understanding the mechanisms that control granule movement to and fusion with the plasma membrane could provide novel targets for improved asthma and allergy therapeutics. To this end, an isolated intact population of granules from the RBL-2H3 mast cell provides a powerful research tool and as such the primary aim of this work was to isolate intact granules from the RBL-2H3 mast cell. Using iso-osmotic Percoll gradients we have isolated an intact granule population from RBL-2H3 mast cells. This granule population contained three granule markers: ??- hexosaminidase, serotonin and chymase. Triton X-100 pre-lysis resulted in loss of granule markers from this main peak, indicating that the isolated granules are in fact intact. Further analysis of the granule population showed that it is free from bulk contamination with other organelles and plasma membrane. The granules were estimated to have a density of 1.055 ??? 1.092g/mL, significantly less dense than that of rat peritoneal mast cell granules (1.2g/mL; [1]). Using an intact versus lysed approach, granule-associated proteins and phosphoproteins, from unactivated RBL-2H3 cells, were determined. Nine unknown granule-associated proteins were found using silver staining of gradient fractions separated on a SDSPAGE gel. In addition, four unknown serine or threonine granule-associated phosphoproteins were found. Molecular weight comparison suggested overlap in some of the unknown proteins and phosphoproteins. Probing for protein kinase C (PKC) isoforms confirmed previous results suggesting that a small population of PKC?? localised to the granules [2], and extended these results to include a population of PKC??I. The serine/threonine phosphatase PP1 does not appear to be granule associated. However, there was a small loss of PP2A from the granules (upon lysis), suggesting that perhaps a subpopulation of PP2A is granule-associated. The main granule peak represents a secretion competent population as Fc??RI-mediated activation of the cells resulted in a significant loss of granule markers from this peak. At the peak rate of antigen-induced secretion a number of changes occur in the phosphorylation of granule-associated phosphoproteins. In addition to an increase in the phosphorylation of three of the phosphoproteins seen in resting mast cell granules, eight new proteins were seen. Whether these proteins are granule-associated is currently unknown. PKC?? was found to translocate away from the granules at the peak rate of secretion, perhaps representing an important control mechanism in granule exocytosis. None of the tested PKC isoforms were found to translocate to the granules, providing little clue as to the identity of the kinase that may be involved in these phosphorylation events. However, as PKC??I is granule-associated and does not translocate off the granules, it would suggest that this kinase might be important for some of the observed phosphorylations. Overall the studies in this thesis show for the first time a rapid gradient-based method for the isolation of intact granules from unactivated and activated RBL-2H3 mast cells. These granules were used to determine granule-associated proteins and phosphoproteins, as well as to investigate changes that occur during the secretory process. In addition, the results show that a number of proteins have increased serine/threonine phosphorylation at the peak rate of antigen-stimulated secretion. This implies that phosphorylation is likely to play a role in the control of granule exocytosis. The identity of these proteins deserves further investigation. Thus, isolated intact RBL- 2H3 mast cell granules provide a powerful research tool to further investigate the mechanism and control of granule exocytosis.