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.

The mitotic spindle checkpoint plays a critical role during the cell cycle by protecting the faithful transmission of chromosomes during mitosis. If chromosomes are improperly bound to the spindle microtubules the checkpoint will prevent progress to anaphase by temporarily arresting cells in metaphase until all the chromosomes are correctly aligned. Bub1b is an essential component of the mitotic spindle checkpoint that transiently localises to kinetochores during mitosis and becomes phosphorylated, a response that is sustained during mitotic arrest. Bub1b has been implicated in other processes related to mitotic progression and is thought to regulate mitotic timing and have a role in caspase mediated cell death after prolonged mitotic arrest. The development of aneuploidy and cancer has been associated with mutations in the BUB1B gene and reduction in the level of Bub1b protein. To further our understanding of Bub1b function in the spindle checkpoint and mitosis, new protein interactions involving Bub1b were identified. This thesis describes the search for alternative proteins that interact with Bub1b, and their function in the mitotic spindle checkpoint and regulation of Bub1b activity. Using a yeast two-hybrid approach, members of the B56 family of regulatory subunits of serine-threonine protein phosphatase (PP2A) were identified as novel interacting partners of Bub1b. Substrate specificity of PP2A is determined by the regulatory subunits. There are five characterised isoforms of the B56 family, each encoded by separate genes. In addition, some isoforms have several recognised splice variants. Confirmation of interactions by alternative methods demonstrated that the isoforms B56γ and B56[epsilon] preferentially interact with phosphorylated Bub1b, whereas the interaction of the remaining B56 isoforms (α, β and [delta]) occurs at a lower affinity with no specificity for the phosphorylated form. It was further demonstrated that B56γ1 associated with phosphorylated Bub1b in vivo. Induced overexpression of splice variants of B56γ1 and B56γ2 demonstrated a significant reduction in levels of phosphorylated Bub1b during mitotic spindle checkpoint activation. In addition, an associated lower mitotic index was evident in cells with B56γ1 overexpression. Specific inhibition of PP2A activity with okadaic acid was shown to prolong Bub1b phosphorylation during normal mitosis and to restore the levels of phosphorylated Bub1b in arrested cells over expressing B56γ. These findings suggest a role for PP2A activity in regulation of Bub1b function that is mediated through substrate recognition by B56 regulatory subunits.

Thesis (M.S.)--University of Missouri--Rolla, 2007. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed December 7, 2007) Includes bibliographical references (p. 28-35).

This thesis examines the interconnection between the in vitro and in vivo phosphorylation of rabbit muscle phosphofructokinase. The first goal of the project was to show whether a novel site of rabbit muscle phosphofructokinase that is subject to in vitro phosphorylation, serine 376, may also become phosphorylated in vivo. Evidence obtained through iron chelate chromatography, amino acid analysis, gas phase sequencing, ammonium sulfate reversed phase high pressure liquid chromatography (HPLC), and matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) spectroscopy of cyanogen bromide digests of the enzyme purified from epinephrine-stimulated rabbit hearts demonstrate the in vivo phosphorylation of serine 376. Parallel experiments with phosphofructokinase isolated from unstimulated rabbit hearts show no detectable phosphorylation of serine 376. The second part of the thesis examines the effects of alterations in experimental conditions on the in vitro phosphorylation of rabbit muscle phosphofructokinase that is catalyzed by the cAMP-dependent protein kinase (cAMP-dPK). Significant phosphorylation of serine 376 takes place in the presence of specific proteins, calmodulin-calcium and troponin C-calcium, that are known to stabilize the catalytically inactive phosphofructokinase dimer. Conditions that stabilize the catalytic activity of phosphofructokinase generally inhibit the in vitro phosphorylation reaction. / Graduation date: 1999

Thesis (Ph. D.)--University of Rhode Island, 2006. / Typescript. Includes bibliographical references (leaves 136-153).

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.

Le Virus de Varicelle et du Zona (VZV) est un -herpesvirus responsable de deux maladies distinctes : la varicelle et le zona. Dans ce travail, nous nous sommes intéressés à la protéine Immediate Early 63 (IE63) du VZV. Cette protéine est essentielle pour la réplication virale et critique pour létablissement de la latence du virus dans les ganglions sensoriels. Lors du cycle lytique, elle présente une localisation majoritairement nucléaire, alors que durant la latence, cest dans le cytoplasme quelle se concentre pour finalement se répartir entre les deux compartiments une fois la réactivation du virus enclenchée. Ses propriétés régulatrices sont sujet à controverse depuis de nombreuses années. Les phénomènes de phosphorylation sont connus pour jouer un rôle clé dans la régulation de lactivité de nombreuses protéines. Dans la première partie de ce travail, nous nous sommes penchés sur la phosphorylation dIE63 par deux types de kinases cellulaires importantes, la Protéine Kinase AMPc-dépendante (PKA) et les Cycline-dépendante Kinases (Cdks). Nous avons tenté de mettre en évidence limpact de cette modification post-traductionnelle sur la localisation cellulaire et les propriétés régulatrices dIE63. Nous avons pu montrer quIE63 est phosphorylée par la PKA. Il sest avéré que cette phosphorylation nétait pas essentielle pour la localisation cellulaire correcte de la protéine, mais bien pour ses propriétés régulatrices. Ensuite, nous avons pu démontrer quIE63 est phosphorylée in vitro par la Cdk1 et la Cdk5, et in vivo par la Cdk1. Cette phosphorylation est apparue comme cruciale pour la localisation cellulaire et lactivité régulatrices dIE63 en cellules Vero. A lentame de la seconde partie de ce travail, une compréhension des fonctions globales dIE63 sur lexpression de lensemble des gènes cellulaires manquait toujours. Dans ce contexte, nous avons décidé dexaminer les effets dIE63 sur la transcription de lensemble du génome cellulaire par la technique de microarray. Nous avons pu montrer que : (i) en labsence dautres protéines virales, IE63 affecte lexpression dun nombre limité de gènes, incluant des gènes impliqués dans la transduction de signaux, la transcription, la réponse immunitaire et la signalisation des protéines « Heat-Shock ». (ii) Lexpression dIE63 provoque suivant les cas une diminution ou une stimulation de la liaison de la RNA polymérase II sur les promoteurs réprimés et activés par la protéine, respectivement. (iii) En cellules HeLa, la phosphorylation correcte dIE63 est cruciale pour ses propriétés régulatrices sur les promoteurs endogènes. Des travaux antérieurs émanant de notre laboratoire ont montré quIE63 était capable dinhiber lexpression de certains gènes dépendants du NF-B comme lIL-8 et lIL-6. De plus, plusieurs études montrent que le VZV est capable déchapper au système immunitaire via notamment une répression des gènes dépendant du NF-B. De manière surprenante, lexpression basale de ces gènes nest pas affectée dans nos conditions, cela étant peut être du à un problème daccessibilité de leur promoteur lié à louverture de la chromatine. De manière à tester cette hypothèse, nous avons mesuré linfluence dIE63 sur lexpression de certains de ces gènes (IL-8, IL-6, ICAM-1 et IB) après un traitement des cellules au TNF, une cytokine proinflammatoire connue pour provoquer louverture de la chromatine au niveau du promoteur de nombreux gènes. Les résultats intéressants obtenus lors de ce travail sont : (i) dans des cellules stimulées au TNF, lexpression de certains gènes dépendant du NF-B est affectée par IE63 dune manière dépendante du promoteur étudié, (ii) le niveau de phosphorylation de la protéine influence ces propriétés régulatrices, (iii) leffet dIE63 est corrélé à une modification de louverture de la chromatine et enfin, (iv) IE63 est capable de modifier la liaison du NF-B sur les promoteurs testés.

Short linear motifs (SLM) play critical roles in cell signaling and are associated with important biochemical events such as phosphorylation, glycosylation, and other post translational modifications. The primary aim of this thesis is to develop a new computational method (“ConDens”) to predict kinase substrates by assessing the evolution of phosphorylation SLM’s in a novel manner. This method could predict yeast Cdc28 kinase substrates that were not confidently detected by several other prediction methods published in literature and was demonstrated to be generalizable to other kinases. Genome-wide predictions experiments with this method also revealed potentially interesting novel substrates of Mec1, Prk1, PKA, and CKII.

Short linear motifs (SLM) play critical roles in cell signaling and are associated with important biochemical events such as phosphorylation, glycosylation, and other post translational modifications. The primary aim of this thesis is to develop a new computational method (“ConDens”) to predict kinase substrates by assessing the evolution of phosphorylation SLM’s in a novel manner. This method could predict yeast Cdc28 kinase substrates that were not confidently detected by several other prediction methods published in literature and was demonstrated to be generalizable to other kinases. Genome-wide predictions experiments with this method also revealed potentially interesting novel substrates of Mec1, Prk1, PKA, and CKII.

Tau protein is one of the microtubule-associated proteins (MAPs) and mainly expressed in neuronal cells. It hasbeen demonstrated that Tau may play an important role in regulating microtubule dynamic in neurons. Structurally and functionally, Tau protein composed of regulatory projection domain in N-terninus and microtubule-binding domain in C-terminus. It has been shown that the biological function of Tau protein was regulated by phosphorylation and dephosphorylation. In Alzheimer¡¦s disease (AD) brain, hyperphosphorylated Tau caused by over active kinases may contribute to the disassociation of Tau from microtubule and form the pathologically hallmarker, paired helical filaments (PHFs). The reason to study cdc2 and GSK3£] is two folds. First, both cdc2 and GSK3£] activities are raised abbrently in AD brain. Second, the phosphorylation sites of cdc2 and GSK3£] have been identified as those in PHFs.These prompted us to regard cdc2 and GSK3£] as candidates that hyperphosphorylated Tau in AD. In the following study, we used immunofluorescence analysis, co-immunoprecipitation and GST-fusion protein pull down assay to clarify the subcellular localization of Tau. We also shown that the interaction between tubulin withfull length Tau (Tau WT) and some Tau mutants that we found that not only Tau WT, but also N-terminus of Tau (Tau-N) and C-terminus of Tau (Tau-C) can bind to tubulin. Surprisingly, we observed that a fragment of N-terminus, Tau 122-244, localized in nucleus. Furthermore, we used tubulin assembly assay to test if tau or its mutants can promote tubulin assembly in vitro. Results showed that only Tau WT can promote tubulin assembly in vitro but not Tau-N or Tau-C. Although Tau-N or Tau-C can bind to tubulin in vivo and in vitro, these mutants did not remain the ability to promote tubulin assembly that suggested both functional domains, N-terminus and C-terminus of Tau, are necessary and essential for the biological function of Tau. On the other hand, we used of phosphorylation assay and site directed mutagenesis to demonstrate that T231 of Tau is one of important phosphorylation sites of cdc2 and GSK3£]. Finally, we used tubulin assembly assay to show that phosphorylated Tau by GSK3£] can negatively regulate the ability of Tau to promote tubulin assembly that indicated that the phosphorylation at T231 may play a role in regulating Tau.