Dissertations / Theses on the topic 'Ser/Thr protein kinases'

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis.

La protéine kinase AMPc-dépendante (PKA) joue un rôle crucial dans la régulation neurohormonale de la fonction cardiaque. L'activation aiguë de la PKA est bénéfique car elle conduit à une augmentation de la contraction cardiaque en phosphorylant les acteurs clés du couplage excitation-contraction. En revanche, son activation chronique est délétère et ces effets semblent faire intervenir la régulation de protéines nucléaires pouvant conduire au remodelage hypertrophique et à l'insuffisance cardiaque. La localisation subcellulaire de la PKA, assurée par des protéines d'ancrage (AKAPs), est importante pour la rapidité et la spécificité d'action des hormones mettant en jeu la voie de l'AMPc. Les niveaux d'AMPc sont régulés par l'activité des adénylate cyclases et des phosphodiestérases (PDEs), et l'état de phosphorylation des protéines cibles de la PKA dépend de l'activité des Ser/Thr phosphatases (PPs). Dans le cœur, les PDEs les plus importantes dégradant l'AMPc sont les PDE3 et les PDE4. Les principales PPs cardiaques sont PP1, PP2A et PP2B. Dans une première partie de mon travail, j'ai mis au point, dans les cardiomyocytes de rats adultes, une mesure de l'activité de la PKA en temps réel dans les compartiments cytoplasmiques et nucléaires. J'ai utilisé pour cela des sondes de type AKAR (A-kinase activity reporters) basées sur le transfert d'énergie de fluorescence (FRET) et localisées spécifiquement dans le noyau ou dans le cytoplasme par des séquences d'adressage ou d'exclusion nucléaires. J'ai ainsi pu montrer qu'une stimulation maintenue des récepteurs β-adrénergiques active la PKA de façon plus importante dans le cytoplasme que dans le noyau, et que cette activation se développe lentement au niveau nucléaire que dans le cytoplasme. De ce fait, une stimulation brève des récepteurs β-adrénergiques active maximalement la PKA dans le cytoplasme, mais de façon marginale dans le noyau. Dans une seconde partie de l'étude, je me suis intéressée au rôle des PDE3 et PDE4 ainsi qu'à celui de PP1, PP2A et PP2B dans la régulation de l'activité PKA cytoplasmique et nucléaire, en réponse à une stimulation β-adrénergique. J'ai montré que la PDE4, mais pas la PDE3, régule l'activité de la PKA cytoplasmique et nucléaire. L'utilisation de souris invalidées pour les gènes Pde4b et Pde4d a révélé que l'isoforme PDE4B est prédominante pour la modulation de l'activité PKA cytoplasmique, alors que les deux isoformes PDE4B et PDE4D contribuent à la régulation de l'activité PKA nucléaire. Finalement, j'ai montré que la PP1 et la PP2A, mais pas la PP2B, participent à la terminaison des réponses β-adrénergiques dans le cytoplasme, alors qu'au niveau nucléaire, la PP1 semble jouer un rôle majeur. En conclusion, ce travail a mis en évidence le rôle des phosphodiestérases et des phosphatases dans l'intégration différentielle des réponses PKA à une stimulation β-adrénergique dans le cytoplasme et le noyau de cardiomyocytes adultes.

Des genes codant pour une famille de ser/thr-kinases chez la cyanobacterie filamenteuse et fixatrice d'azote anabaena sp. Pcc 7120, ont ete isoles dans notre laboratoire. Ces genes sont impliques dans la croissance ou developpement cellulaire. Les substrats de cette famille de ser/thr-kinases sont encore inconnus. Un des substrats potentiels de ces kinases est la proteine pii, connue pour etre phosphorylee sur un residu ser. Cette proteine est impliquee dans la regulation de l'assimilation de l'azote, et dans la coordination des metabolismes azote et carbone. Le gene glnb codant pour pii a ete clone chez anabaena sp. Pcc 7120. La cinetique de modification de pii pendant le developpement des heterocystes est de facon dynamique, ce qui incite a poursuivre l'etude du role eventuel de pii dans la differenciation cellulaire induite par la carence en azote combine. L'une des ser/thr-kinases de cette bacterie, pknc, peut phosphoryler la proteine pii recombinante sur un residu ser in vitro. L'introduction d'une mutation dans le domaine catalytique elimine l'activite de proteine-kinase de pknc. La phosphorylation de pii par pknc est stimulee par l'atp, mais l'-cetoglutarate, glutamate ou glutamine n'ont pas d'effet sur le niveau de phosphorylation de pii in vitro. Un essai de mutagenese suggere que pii est probablement essentielle chez anabaena sp. Pcc 7120. Afin de dissequer la fonction biologique de pii, nous avons construit des mutants ponctuels sur le residu ser qui constitue la cible de phosphorylation. Les resultats d'analyses seront presentes. L'inactivation du gene pknc n'influence pas le niveau de phosphorylation de pii chez anabaena sp. Pcc 7120. De nombreuses observations experimentales suggerent que pii joue des roles differents chez les souches unicellulaires non fixatrices d'azote et chez les souches filamenteuses fixatrices d'azote. Plusieurs hypotheses seront discutees afin de comprendre et d'expliquer le role de pii et pknc chez anabaena sp. Pcc 7120.

Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.

Mycobacterium tuberculosis, le pathogène responsable de la tuberculose, a la capacité d’évader le système immunitaire et rester en état dormant pendant des années pour resurgir suite à un affaiblissement du système immunitaire. Pour comprendre les réponses de la mycobactérie aux changements dans l’environnement de l’hôte, l’étude des protéines régulatrices impliquées dans la transduction des signaux est d’une importance fondamentale. Dans ce contexte, mon projet de thèse visait à étudier la structure et fonction de la Ser/Thr kinase essentielle PknB et de la Ser/Thr phosphatase PstP, afin de comprendre leur rôle dans la signalisation chez M. Tuberculosis et d’identifier des cibles potentielles pour le développement de nouvelles thérapies. Un substrat a été identifié pour PknB et les études d'interaction protéine-protéine ont permis de proposer un modèle de recrutement du substrat via la boucle d'activation de la kinase. Des criblages d'inhibiteurs potentiels ont identifié plusieurs composés qui ont une activité inhibitrice in vitro et sur des cultures de mycobactéries, et les structures de complexes PknB-inhibiteurs ont été déterminées. La caractérisation structurale et fonctionnelle des Ser/Thr phosphatases PstP de M. Tuberculosis et MsPP de M. Smegmatis, a mis en évidence un centre métallique trinucléaire avec des affinités variables pour les métaux, et un possible rôle dans la fixation du substrat pour le troisième métal. Des complexes à résolution atomique de MsPP montrent 3 possibles étapes le long du cycle catalytique, qui nous a permis de proposer un mécanisme de type acide/base pour l'hydrolyse du phosphate, qui passerai par un état intermédiaire associatif.

Protein kinase CK2 and CK1 are a pleiotropic and evolutionary conserved serin-threonin kinase that is involved in several cellular processes. A number of studies revealed many mechanisms through which this kinase regulates cell cycle, apoptosis, cell survival and tumorigenesis. CK2 participates in many developmental pathways, of which particularly relevant for hemo-lymphopoiesis are those dependent on Hedgehog, NF-κB and STAT3, which regulate cell differentiation, proliferation, self-renewal as well as lineage choice commitment.. CK1 regulates also molecular pathways which are important for multiple myeloma plasma cells survival, like WNT/β-catenin pathway and PI3K/AKT pathway. However, despite all this data, little is known about the role of CK2 and CK1 in B-lymphopoiesis and lymphomagenesis. To elucidate the physiological and pathogenetic role of CK2 and CK1 in B-lymphocytes, we generated B cell specific conditional KO mice, were we studied the effects of deletion during normal B cell development with multiparameter flow cytometry analysis. In the bone marrow (BM), CK2β KO mice displayed a reduction of B cells, especially of the recirculating population of transitional and follicular (FO) B-cells. In peripheral blood and spleen the number of B-cells was markedly reduced. In the spleen of CK2β KO we observed an imbalance between the amount of FO and marginal zone (MZ) B-cells was found with an absolute reduction of FO B cells by approximately 2-folds and an increase of MZ B-cells and MZB cell precursors by up to three folds.. In vitro class-switch recombination assays demonstrated impairment in IgG1 and IgG3 class-switch and a marked reduction of the generation of antibody-producing cells. In CK1 KO mice we observed the totally absence of mature B cells and the presence of early precursors B cells. CK1 HET mice showed a reduction of B cells in bone marrow and an light imbalance of FO B cells an MZB cells in spleen. In vitro class-switch recombination assays doesn’t showed significant difference between HET and CTRL mice in IgG1 and IgG3 class-switch. Here, we found that the β subunit of protein kinase CK2 is a novel regulator of peripheral B cell differentiation. CK2β has a role in regulation of the GC reaction and in homeostasis of FOB and MZB cells. Furthermore we found that CK1 has a pivotal role in early B cells development. On one side our data enrich the knowledge on the mechanisms regulating B-cell development, on the other side they inform about the potential mechanisms altered by CK2 and CK1 during B-cell tumorigenesis.
Protein kinase CK2 and CK1 are a pleiotropic and evolutionary conserved serin-threonin kinase that is involved in several cellular processes. A number of studies revealed many mechanisms through which this kinase regulates cell cycle, apoptosis, cell survival and tumorigenesis. CK2 participates in many developmental pathways, of which particularly relevant for hemo-lymphopoiesis are those dependent on Hedgehog, NF-κB and STAT3, which regulate cell differentiation, proliferation, self-renewal as well as lineage choice commitment.. CK1 regulates also molecular pathways which are important for multiple myeloma plasma cells survival, like WNT/β-catenin pathway and PI3K/AKT pathway. However, despite all this data, little is known about the role of CK2 and CK1 in B-lymphopoiesis and lymphomagenesis. To elucidate the physiological and pathogenetic role of CK2 and CK1 in B-lymphocytes, we generated B cell specific conditional KO mice, were we studied the effects of deletion during normal B cell development with multiparameter flow cytometry analysis. In the bone marrow (BM), CK2β KO mice displayed a reduction of B cells, especially of the recirculating population of transitional and follicular (FO) B-cells. In peripheral blood and spleen the number of B-cells was markedly reduced. In the spleen of CK2β KO we observed an imbalance between the amount of FO and marginal zone (MZ) B-cells was found with an absolute reduction of FO B cells by approximately 2-folds and an increase of MZ B-cells and MZB cell precursors by up to three folds.. In vitro class-switch recombination assays demonstrated impairment in IgG1 and IgG3 class-switch and a marked reduction of the generation of antibody-producing cells. In CK1 KO mice we observed the totally absence of mature B cells and the presence of early precursors B cells. CK1 HET mice showed a reduction of B cells in bone marrow and an light imbalance of FO B cells an MZB cells in spleen. In vitro class-switch recombination assays doesn’t showed significant difference between HET and CTRL mice in IgG1 and IgG3 class-switch. Here, we found that the β subunit of protein kinase CK2 is a novel regulator of peripheral B cell differentiation. CK2β has a role in regulation of the GC reaction and in homeostasis of FOB and MZB cells. Furthermore we found that CK1 has a pivotal role in early B cells development. On one side our data enrich the knowledge on the mechanisms regulating B-cell development, on the other side they inform about the potential mechanisms altered by CK2 and CK1 during B-cell tumorigenesis.

Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第6788号
医博第1888号
新制||医||664(附属図書館)
15860
UT51-97-H172
京都大学大学院医学研究科分子医学系専攻
(主査)教授 月田 承一郎, 教授 永田 和宏, 教授 中西 重忠, 教授 成宮 周
学位規則第4条第1項該当

En aquesta tesi s'apliquen eines bioinformàtiques a l'estudi de determinats sistemes biològics. En particular, l'estudi teòric de la influència de determinats aminoàcids sobre l'estructura i la dinàmica dels elements d'estructura secundària de les proteïnes s'aplica a la modelització per homologia dels receptors acoblats a proteïna G (GPCRs) i a l'estudi dels seus mecanismes d'activació.
Se sap que determinats residus, com prolina, serina o treonina, provoquen distorsions locals en l'estructura de les hèlices a. L'anàlisi de bases de dades de seqüències de segments transmembrana mostra com certes combinacions d'aquests residus són més comunes que d'altres, i que algunes d'elles estan sobre-representades de manera significativa, mentre que d'altres estan clarament sots-representades. La restricció d'aquesta anàlisi de seqüències a la regió transmembrana dels GPCRs de la Classe A mostra com aquestes combinacions es troben en posicions específiques i, a més, es troben conservades en certes subfamílies de receptors.
L'estructura i la dinàmica de les hèlices transmembrana que contenen aquestes combinacions de prolina i serina o treonina s'han estudiat mitjançant simulacions de dinàmica molecular en un entorn hidrofòbic explícit. Els resultats mostren com algunes d'aquestes combinacions indueixen distorsions importants en l'estructura de l'hèlix a, degut al seu efecte desestabilitzador de la xarxa de ponts d'hidrogen que dóna estabilitat a l'hèlix.
Aquests resultats s'han aplicat a la construcció d'un model tridimensional del receptor de quimiocines CCR5 , utilitzant tècniques de modelització molecular per homologia. En aquest model es proposa que les hèlices transmembrana (TMH) 2 i 3 del receptor CCR5 són estructuralment diferents del patró de rodopsina. TMH2 està més doblegada degut a la presència d'un motiu Thr-X-Pro, que, a més, fa que aquesta hèlix es doblegui cap a TMH3. Així doncs, es proposa que, en aquest receptor, aquestes dues hèlices interaccionen. Aquesta interacció estaria mediada per la presència de residus hidrofòbics conservats i específics en les dues hèlices. Aquestes hipòtesis han estat posades a prova mitjançant experiments de mutagènesi dirigida, gràcies a la col·laboració amb l'Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire (IRIBHN), Université Libre de Bruxelles. Els resultats experimentals permeten establir la hipòtesi que la interfície TMH2-TMH3 participa en l'activació induïda per quimiocines del receptor CCR5.
Com a conclusió, aquesta tesi pretén mostrar com, mitjançant la utilització d'eines bioinformàtiques, és possible traduir les seqüències primàries de proteïnes i les interaccions a nivell atòmic en estructures tridimensionals de proteïnes. A més, aquesta tesi mostra que, encara que l'estructura tridimensional de la rodopsina bovina és un patró útil per la modelització per homologia de GPCRs, s'han de tenir en compte de manera explícita les especificitats de seqüència de cada receptor per tal de construir models de receptors particulars. Aquestes especificitats de seqüència consisteixen en patrons de seqüència conservats en determinades famílies, que es tradueixen en divergències estructurals. Entre aquests patrons de seqüència, es proposa que els residus de serina i treonina, sols o combinats amb residus de prolina propers, poden modular la geometria de les TMHs, degut a la seva capacitat d'interferir amb la xarxa de ponts d'hidrogen que dóna estabilitat a les hèlices a.
Finalment, es proposa que la influència dels motius de serina, treonina i prolina en l'estructura de les TMHs pot estar relacionada amb els processos d'activació dels GPCRs de la Classe A i, possiblement, d'altres proteïnes de membrana. En els GPCRs, aquests motius poden haver evolucionat per tal d'adaptar uns mecanismes d'activació conservats als lligands característics de cada família de receptors.
This thesis is framed in the study of particular biological systems through the use of bioinformatics. In particular, the theoretical study of the influence of certain amino acids on the structure and dynamics of the secondary structure elements of proteins has been applied to homology modelling of G protein-coupled receptors (GPCRs) and to the study of their mechanisms of activation.
Certain residues, as proline, serine or threonine, are known to induce local distortions in the a-helical structure. Analysis of sequence databases of transmembrane segments evidence that certain combinations of these residues are more common than others, and that some of them are significantly over-represented, while others are clearly under-represented. The focusing this sequence analysis on the transmembrane region of Class A GPCRs illustrates that these combinations are located in some specific locations and conserved within certain subfamilies of receptors.
The structure and dynamics of transmembrane a-helices containing these combinations of proline and serine or threonine have been studied using molecular dynamics simulations in an explicit hydrophobic environment. The results show how some of these combinations induce significant distortions in the a-helical structure, due to their effect on the hydrogen bond network that stabilizes the helix.
These results have been applied to the building of a three-dimensional model of the chemokine CCR5 receptor, using homology modelling techniques. In this model, transmembrane helices (TMH) 2 and 3 of CCR5 are proposed to be different from the bovine rhodopsin template. TMH2 is more bent due to the presence of a Thr-X-Pro motif, which, in turn, induces this helix to lean towards TMH3. As a consequence, an interaction between these two helices is proposed for this particular receptor. This interaction would be mediated through the presence of specific and conserved hydrophobic and aromatic residues in both helices. These hypothesis have been tested through site-directed mutagenesis experiments, thanks to a collaboration with the Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire (IRIBHN), Université Libre de Bruxelles. The experimental results let us to hypothesize that the TMH2-TMH3 interface is involved in the chemokine-induced activation of the CCR5 receptor.
As a conclusion, this thesis aims to show how through the use of bioinformatics tools, primary sequences of proteins and interactions at an atomic level can be translated to three-dimensional protein structures. In addition, this thesis illustrates that, even though the three-dimensional structure of bovine rhodopsin is a very useful template for homology modelling of GPCRs, the sequence specificities of each receptor have to be explicitly taken into account in order to build models. These sequence specificities consist in sequence patterns conserved within certain families, which are translated into structural divergences. Among these sequence patterns, we hypothesize that serine and threonine, alone or combined with nearby proline residues, can modulate the geometry of TMHs, due to its capability to interfere with the hydrogen bond network that stabilize a-helices.
Finally, we propose that the influence of serine, threonine and proline motifs in the structure of TMHs may be related to processes of activation in the Class A of GPCRs, and, possibly, other membrane proteins as well. In GPCRs, these motifs may have evolved in order to adapt a conserved mechanism of activation of the G protein to the cognate ligands of each receptor family.

The Protein kinase C (PKC) superfamily is a key regulator in platelet activation with individual isoforms playing distinct roles. This thesis focuses on the role of the novel PKC isoforms downstream of several agonists using both pharmacological and genetic approaches and human and mouse platelets. Quantification of the protein levels of PKC isoforms identified different levels of the five major PKC isoforms expressed in human platelets and also differences between levels of the same isoform in human and mouse platelets. Use of a selection of broad spectrum and isoform-specific inhibitors, identified both positive and negative novel roles for PKC in the regulation of human and mouse platelets. A net positive role for PKC was found in GPVI, Clec-2, and PAR receptor signalling, with classical isoforms of PKC playing a major role in aggregation and dense granule secretion. A novel negative regulatory role was also identified in the regulation of ADP-induced platelet activation for PKC~, and both PKCE and PKC~ in human and mouse platelets respectively. Gene knock-out mouse models confirmed a positive regulatory role for PKCe in allb~3 outside-in signalling but identified no other regulatory role for PKCe in agonist induced platelet activation. Despite this relatively minor role, functional redundancy was identified between PKCe and PKCE isoforms in haemostasis, as tail bleeding was significantly increased in mice deficient in both novel isoforms. The work presented here identifies key roles for the PKC superfamily in the complex regulation of platelet activation, with different isoforms supporting and limiting the process of thrombus formation and haemostasis.

L’un des obstacles majeurs au développement de nouveaux antipaludiques est notre connaissance limitée de la biologie parasitaire et la rareté des cibles thérapeutiques potentielles identifiées. Les interactions protéines-protéines sont impliquées et essentielles dans divers processus biologiques incluant les modifications post-traductionnelles. Les interactions substrats-kinases ou phosphatases sont considérées comme une liaison transitoire et jouent un rôle central et essentiel dans le cycle cellulaire de Plasmodium. La Ser/Thr Protéine Phosphatase de Type 1 (PP1), l’une des phosphatases majeurs des eucaryotes, est essentielle à la survie du parasite Plasmodium falciparum, responsable du paludisme. Elle est régulée par diverses sous-unités régulatrices dont plus de 200 ont été identifiées chez l’Homme, mais seulement 4 chez Plasmodium.Afin d’explorer le réseau de régulation de la P. falciparum PP1 (PfPP1), nous avons utilisé trois approches complémentaires pour caractériser l’interactome de la PfPP1. La purification par co-affinité suivie d'une analyse par spectrométrie de masse a identifié 6 protéines interagissant avec la PfPP1 dont 3 contenaient le motif consensus d’interaction RVxF, 2 autres le motif Fxx[RK]x[RK], également connu pour interagir avec la phosphatase et une protéine avec les deux motifs de liaison. Le criblage par double hybride chez la levure a identifié 134 protéines dont 30 présentent le motif RVxF et 20 ont le motif de liaison Fxx[RK]x[RK]. Le criblage in silico du génome de P. falciparum en utilisant une séquence consensus du motif RVxF a révélé 55 partenaires potentiels de la PfPP1. Afin de confirmer l’interaction de certaines protéines, 35 partenaires candidats ont été validés par un test d’interaction de type ELISA. Les résultats ont permis de détecter aussi bien des partenaires conservés de la PP1 qu'un nombre élevé d'interacteurs spécifiques à la PP1 du parasite et montrent une grande diversité dans les fonctions biologiques impliquant la PP1 chez Plasmodium. Parmi ces candidats, un partenaire appelé Gametocyte EXported Protein 15 (GEXP15) a été confirmé comme un réel partenaire de la PfPP1 par différentes approches. De plus, GEXP15 est surexprimé chez les gamétocytes, stade responsable de la transmission du parasite chez le moustique. Ces résultats ainsi que des études fonctionnelles seront présentés
A major obstacle in the development of novel anti-malarials is our limited knowledge of basic parasite biology and the paucity of identified potential intervention targets. Protein-protein interactions are involved and essential in broad range of biological processes including the post-translational modifications. Substrate-kinase or phosphatase interactions are considered as transient binding and play a central and essential role in Plasmodium cell cycle. The Ser/Thr Protein Phosphatase Type 1 (PP1), one of the main contributors of eukaryotic phosphatase activity, is essential to malaria parasite Plasmodium falciparum and is highly regulated by many regulatory subunits. In humans, there are about 200 distinct regulators, however, only 4 have been so far reported in Plasmodium.To explore the P. falciparum PP1 (PfPP1) regulatory network as complete as possible, we carried out three complementary approaches to characterize the PfPP1 interactome. Co-affinity purification followed by Mass Spectrometry-based proteomics identified 6 PfPP1 interacting proteins (PIPs) of which 3 contained the RVxF consensus binding motif, 2 PIPs with a Fxx[RK]x[RK] binding motif, one with both binding motifs. The Yeast Two-Hybrid screening identified 134 proteins of which 30 have the RVxF binding motif and 20 contain the Fxx[RK]x[RK] binding motif. The in silico screen of the P. falciparum genome using a consensus RVxF motif as template revealed the presence of 55 potential PfPP1 interacting proteins. As further demonstration, 35 candidate partners were validated in an independent ELISA-based assay using recombinant proteins. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1, indicating a high diversity of biological functions for PP1 in Plasmodium. Among these candidates, one partner assigned as Gametocyte EXported Protein 15 (GEXP15) has been confirmed as a direct interactor of PfPP1 by different approaches. In addition, GEXP15 is over-expressed during gametocyte stage, responsible for the transmission of the parasite in the mosquito. These results as well as functional studies will be presented and discussed

Notre laboratoire a précédemment proposé un concept appelé "Drug phosphatase Technology" (DPT) basé sur l'utilisation de séquences pénétrantes cationiques capables d'interagir avec les phosphatases PP1/PP2A pour déréguler spécifiquement des signaux intracellulaires. Les Ser. Thr protéine-phosphatases PP1 et PP2A sont des acteurs clé de la signalisation cellulaire et la dérégulation de leur activité via leur interaction avec des partenaires d'origine cellulaire ou virale conduit souvent à de graves dysfonctionnements. Par exemple, la protéine E4orf4 des adénovirus interagit avec PP2A1 et entraîne l'apoptose des cellules infectées ou transformées alors que les cellules saines ne sont pas affectées. Lors de cette étude nous avons identifié une séquence d'interaction entre E4orf4 et PP2A1 à partir de laquelle nous avons caractérisé le peptide DPT-E4orf44, qui provoque l'apoptose de certaines cellules tumorales humaines sans affecter les cellules saines de type fibroblastes. Les protéine-phosphatases de la famille PP1 et PP2A jouent également un rôle important dans la régulation physiologique des substrats neuronaux Tau et APP (précurseur de peptide amyloïde). La diminution de l'activité phosphatase dans les cellules nerveuses entraine une augmentation de l'état de phosphorylation de ces substrats chez les patients atteints de la maladie d'Alzheimer. La deuxième partie de ce travail de thèse a permis de mettre en évidence l'implication les protéine-phosphatases PP1 et PP2A dans la régulation de la phosphorylation du résidu T668 d'APP dont la modification post-traductionnelle est essentielle pour la maturation et le clivage de l'APP
OuTlabhas previousry proposed a concept denominated "Drug Phosphatase Technology" (DFT) based on the use of cationic penetrating sequences capable of interacting with PP1/PP2A phosphatases in order to deregulate specific intracellular signais Ser/Thr proteins-phosphatases of PP1 and PP2A family are key factors of the cellular signalization and deregulation of their activity through interaction with cellular or viral protein often leads to severe dysfunctions. For exemple, E4orf4 protein of adenoviruses interacts with PP2A1 and leads to apoptotic death in infected or transformed cells while healthy cells remain unaffected. In a first part of this work, we have identified an interacting sequence between PP2A1 and E4orf4 from which we - have characterized the peptide DFT-F4orf44, which provokes the apoptosis of a subset of human tumoral cellswithout affecting healthy cells of fibroblastic type. Protein-phosphatases of PP1 and PP2A family also play a crucial role in the physiological regulation of neuronal substracts Tau and APP (Amylold Peptid Precursor). A diminution of the phosphatase activity in nerve cells leads to a drastically increasjng of phosphorylation state of these proteins for patients affected by Alzheimer's disease. I The second part of this work has allowed the identification of the phosphatase proteins responsible for the regulation of phosphorylation of APP T668 residue, which is an essential modification for APP maturation and processing

Afin de déjouer les défenses immunitaires de l’hôte et créer les niches nécessaires à leur survie, les bactéries pathogènes mettent on œuvre de nombreux mécanismes ciblant les voies de signalisation de la cellule hôte. L’un de ces mécanismes repose sur la sécrétion de protéines bactériennes dans les cellules cibles afin de moduler directement leurs réseaux de signalisation. Cependant, les signaux, les senseurs et les effecteurs impliqués dans ces régulations sont encore peu ou mal connus. La détection de l’environnement dans la cellule hôte lors de l'infection est l’élément clé d’une réponse adaptée, et les systèmes de signalisation basés sur les mécanismes de phosphorylation sont indispensables à l'adaptation hôte-pathogène. L’aspect innovant de ce projet repose sur l’étude du rôle des Ser/Thr kinases et phosphatases sécrétées lors des interactions hôte-pathogène, modifiant ainsi la réponse globale de l’hôte durant l’infection. Pendant ma thèse, j’ai tout d’abord étudié le rôle d’une nouvelle protéine kinase bactérienne identifiée chez Coxiella burnetii, nommée CstK (Coxiella serine threonine Kinase). C. burnetii, l’agent étiologique de la zoonose appelée fièvre Q, modifie les défenses de la cellule hôte, permettant sa réplication dans des vacuoles spécifiques à l’intérieur de la cellule hôte. Par ailleurs, la sécrétion d’un grand nombre d’effecteurs bactériens est indispensable au détournement du phagosome par Coxiella. Nous ainsi avons démontré que cette potentielle protéine kinase, identifiée in silico dans le génome de C. burnetii, est capable de s’autophosphoryler et par conséquent possède une activité kinase. De plus, nous avons identifié différentes protéines spécifiques de la cellule hôte interagissant avec CstK à l'aide du modèle amibe Dictyostelium discoideum, un phagocyte professionnel eucaryote, permettant des études génétiques et biochimiques. Dans la deuxième partie de mon projet, je me suis intéressée au rôle d’une probable protéine sécrétée, la tyrosine phosphatase PtpA, durant l’infection par Staphylococcus aureus. Bien connue dans les hôpitaux, où elle est responsable de nombreuses maladies nosocomiales, cette bactérie possède un grand nombre de facteurs de virulence, responsables d’infections variées, et l’apparition exponentielle de souches multi-résistantes en font un problème majeur. Ce pathogène est capable d’envahir et de persister dans un grand nombre de types cellulaires différents chez l’Homme, en sécrétant des protéines effectrices qui vont moduler les réponses cellulaires. Nous avons démontré que PtpA était sécrétée durant la phase de croissance bactérienne, et pu déterminer que PtpA possédait une activité tyrosine phosphatase, régulée par la tyrosine kinase CapA1B2 de S. aureus. Enfin, en utilisant le modèle D. discoideum, nous avons pu identifier des protéines de l’hôte qui interagissent avec PtpA, mais leur rôle dans l’infection n’est pas encore connu
Bacterial pathogens have developed diverse strategies towards host signalling pathways, in order to subvert the immune response and/or create permissive niches for their survival. One such strategy is based on the secretion of bacterial signalling proteins into the target host cells, thereby directly modulating the status of host signalling networks. Because the mechanisms involved are largely intractable to most in vivo analyses, very little is known about the signals, sensors, and effectors mediating these adaptations. Sensing the host environment is a key component to execute appropriate developmental programs, and the eukaryotic-like phosphosignaling systems in prokaryotes are emerging as equally important regulatory systems as the well-known eukaryotic systems, but the study of their functions is still in its infancy. The innovative aspect of this project resides in the study of the emerging role of secreted Ser/Thr kinases and phosphatases in the control of host-pathogen interactions thus modifying the global host response during infection. During my thesis, I first investigated the role of a novel bacterial protein kinase identified in Coxiella burnetii that we named CstK (Coxiella serine threonine Kinase). C. burnetii, the etiological agent of the emerging zoonosis Q fever, subverts host cell defenses, permitting its intracellular replication in specialized vacuoles within host cells. Secretion of a large number of bacterial effectors into host cell is absolutely required for rerouting the Coxiella phagosome. We demonstrated that this putative protein kinase identified by in silico analysis of the C. burnetii genome is able to autophosphorylate and undergoes in vitro phosphorylation. Moreover, we identified specific host cell proteins interacting with CstK, by the use of the model amoeba Dictyostelium discoideum, an eukaryotic professional phagocyte amenable to genetic and biochemical studies. In the second part of my project, I was interested in the role of a putative secreted protein tyrosine phosphatase (PtpA) during Staphylococcus aureus infection. Well-known in hospital-acquired diseases, this bacteria produces multiple virulence factors that lead to various severe diseases, and the increase of multi-resistant strains is a major concern. This pathogen has the ability to invade and persist in a number of different human host cell types, secreting effector proteins to modulate cellular responses. Here we demonstrated that PtpA is secreted during the bacterial growth. We also determined that PtpA presents a tyrosine phosphatase activity that is regulated by the tyrosine protein kinase CapA1B2 of S. aureus. At last, using the D. discoideum model, we identified some host proteins that interact with PtpA, but their link with infection still remain to be studied

Regulatory protein phosphorylation is a well conserved mechanism of signal transduction in all biological systems. In bacteria, signal transduction by phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on serine (Ser) and/or threonine (Thr) residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such as E. coli, which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Bacterial Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C-type phosphatases are referred to as eukaryotic-like Ser/Thr phosphatases (eSTPs). eSTPs have been identified in a number of bacteria, but none have been reported in E. coli. The work presented in this thesis was aimed at identifying and biochemically characterizing a eukaryotic-like Ser/Thr phosphatase and its partner Ser/Thr kinase in E.coli. Chapter 3 describes the identification of a novel PP2C-like Ser/Thr phosphatase PphC encoded by an E. coli ORF, yegK, and characterization of its biochemical properties including kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. I investigated differences in the activity of this protein in closely related E. coli strains. Finally, I demonstrated that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon suggesting that they most likely function as a pair in regulating Ser/Thr phosphorylation. Chapter 4 describes the biochemical characterization of a Ser/Thr kinase YegI in E. coli. I show that YegI is an active kinase with significant structural homology to eukaryotic Ser/Thr kinases. The YegI kinase domain is tethered to a cytoplasmic C-terminal domain containing two non-specific DNA binding Helix-hairpin helix motifs. I have identified enolase and elongation factor-Tu (EF-Tu) as potential physiological substrates of YegI and have demonstrated that phosphorylation of EF-Tu by YegI inhibits protein translation in vitro.

Reversible protein phosphorylation is considered the universal language for intracellular communication in all living organisms. This process, catalysed by protein kinases and phosphatases, enables the translation of extracellular signals into cellular responses and also allows for adaptation to a constantly changing environment. In recent years, a number of bacterial eukaryotic-type Ser/Thr protein kinases and phosphatases have been identified. However, their precise functions and substrates are not yet well defined. The genome of opportunistic human pathogen Pseudomonas aeruginosa contains at least five genes encoding putative eukaryotic-type Ser/Thr protein kinases and phosphatases. In the first part of this study, we have attempted to establish the role of Ser/Thr protein kinase PpkA and phosphatase PppA, which belong to type VI secretion system H1-T6SS. Double mutant strain ∆pppA-ppkA was prepared in P. aeruginosa PAO1 background. Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pigment pyocyanin. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the...

Streptococcus pneumoniae encodes single serine/threonine protein kinase StkP and its cognate protein phosphatase PhpP. This signalling couple phosphorylates/dephosphorylates many target proteins involved in various cellular processes. So far, only few ot them was characterized in detail. Global phosphoproteomic analysis in the ∆stkP mutant strain background resulted in the identification of protein Spr0175 as phosphorylated on threonine 7. The main aim of this work was to characterize this new substrate. The ∆spr0175 mutant strains were prepared in the wild type genetic background Rx and R6 and then monitored for their growth and cell morphology. Mutant strains exhibited morphological defects revealing potential involvement of Spr0175 in the process of cell division. In the wild type D39 the deletion was unsuccesful, which may entail possible essentiality of Spr0175 in D39 strain. The results obtained also confirmed that the Spr0175 is modified in in vitro and in vivo conditions at threonine 7. In vitro study also confirmed minor phosphorylation at T4 residue. By using co-immunoprecipitation assay we demonstrated that Spr0175 protein can form oligomeric structures. Another aim of this work was cellular localization of Spr0175. By using fluorescent microscopy we showed that GFP-Spr0175 fusion...

Protein phosphorylation by protein kinases is a key mechanizm that enables both eukaryotic and prokaryotic organizm sense and read environmental signals and convert these signals into changes in gene expression and thus proper biological response. One of the main phosphorylation systems in bacteria consists of eukaryotic-like Ser/ Thr protein kinases. The genome of human pathogen Streptococcus pneumoniae contains single Ser/ Thr protein kinase StkP. StkP regulates virulence, competence, stress resistance, gene expression and plays an important role in the regulation of cell division cycle. Analysis of phosphoproteome maps of both wild type and ΔstkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including the cell division protein DivIVA (NOVÁKOVÁ et al., 2010). DivIVA in S. pneumoniae is localized at midcell and at the cell poles. It was proposed to be primarily involved in the formation and maturation of the cell poles (FADDA et al., 2007). The aim of this thesis was to investigate phosphorylation of the cell division protein DivIVA in S. pneumoniae. Gene divIVA was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of DivIVA by StkP was examined in a kinase assay. We confirmed that DivIVA is a direct...

RdoA is a eukaryotic-like serine/threonine protein kinase found in Salmonella typhimurium. It is a downstream effector of the Cpx stress response pathway and has been phenotypically characterized to have a functional role in flagellin phase variation and long-term bacterial survivability. Structurally, RdoA is homologous to, choline kinase and aminoglycoside (3’) phosphotransferase IIIa (APH[3’]IIIa). These kinases all belong to a protein kinase superfamily and share highly conserved residues/motifs in their catalytic domain. In RdoA seven of these conserved amino acids were proposed to have functional roles in the phosphotransfer mechanism. Mutation of these proposed catalytic domain residues resulted in a loss of in vitro kinase activity and in vivo RdoA function for a majority of the mutants. Four of the mutants also exhibited decreased levels of stable RdoA compared to wildtype. Many protein kinases regulate activity through phosphorylation of an activation loop. Although RdoA does not contain a canonical activation loop, its carboxyl terminus is proposed to play a similar regulatory function. Mutations of a putative autophosphorylation target in the carboxyl terminus resulted in loss of in vitro kinase activity. Truncations of this region also resulted in loss of kinase activity, as well as decreasing RdoA stability. The length of the carboxyl terminus in the kinase was shown to be an important determinant in the overall structural stability of RdoA. Mutational analyses of conserved amino acid residues surrounding the putative substrate-binding cleft of RdoA revealed site specific mutants with diminished in vitro phosphorylation activity and/or RdoA levels. A subset of these mutants for which no in vitro kinase activity was detected were still able to complement RdoA function in vivo. Taken together these results indicate that this region of the protein is important for RdoA function. In summary, this work has generated a panel of RdoA mutants with several unique phenotypes that will facilitate characterization of RdoA function and of regions of the protein
Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-29 21:35:42.815

Protein kinases, the third most populous protein family, are a major class of enzymes that regulate a wide range of cellular processes by phosphorylating multiple cellular proteins. There are many conserved sequence motifs within the catalytic domain that are essential for the regulation of the proteins, and the catalytic core is commonly shared across all typical protein kinases. In this thesis, we present identification and comprehensive analyses of Ser/Thr or Tyr (STY) protein kinases encoded in many pathogenicity causing organisms. The role of kinases in causing pathogenicity has been elucidated in many of the previous studies. With the emergence of diverse pathogenic species in the last few decades, the protein kinases of the pathogens and the respective hosts might have evolved to adapt to the hostile environment and tolerate stress response. Hence, it is essential to even perform a comparative study of kinases within pathogens against those encoded in their host species. Protein kinases can be identified using sequence-based approaches. However, some of the kinase subfamilies have highly diversified, and one requires sensitive homology detection approaches for their identification. Therefore, we developed a fresh protocol named "Master Blaster" for the detection of distantly related proteins. The performance of Master Blaster is evaluated by comparing against widely-used profile based and hidden Markov model-based methods. An improvement in fold coverage using Master Blaster is reported. We also used artificially designed sequences for detecting distantly related proteins using Master Blaster. Use of the designed sequences was found to be useful in connecting protein families that are highly diverged at sequence level. We applied the developed protocol to identify protein kinases encoded in Candida albicans and performed an extensive analysis of these kinases using sequence-based methods. A comparative study of kinases within C. albicans, pathogenic and non-pathogenic non-albicans Candida species, Baker's yeast and Human was performed. This study has resulted in identifying organism-specific kinases and signature motifs in kinases of pathogenicity causing Candida species. Domain architectures of kinases are also found to be organism-specific. Some of the protein kinases within industrial relevant Candida species such as Pseudozyma antarctica are found to be similar to those encoded in human. Kinases within each subfamily can recognize and phosphorylate multiple substrates and the substrates that are recruited could be specific to a particular kinase subfamily based on their functional role. Phosphorylation of specific substrates is possible due to the conserved substrate binding residues within in each kinase subfamily. Most of the kinase classification schemes depends on the conservation within the catalytic domain region and does not take into account the conservation within the substrate binding regions. This could lead to misclassification of some of the kinases. Hence, we proposed a new approach to classify kinases based on conservation of their substrate binding residues. Using the proposed scheme, we could identify signature regions within some of the kinase subfamilies and these regions can be used for the re-classification of protein kinases. In this thesis work, we also identified kinases encoded in viral genomes using rigorous sequence-based methods and reported the sequence similarities and differences between kinases within various viral genomes. Kinases are detected in viruses having double stranded DNA genome and in some of the subfamilies of retroviruses. In none of the host infecting viruses, kinases could be detected. The putative protein kinases from giant viruses are found to be similar to the retroviral oncogenic kinases. The substrate binding regions within some of the viral kinases are identified using the new classification scheme. This work can be extended by applying developed protocols for studying protein kinases encoded in different organisms.

ANGLICKÝ ABSTRAKT Functional study of the putative nucleotidase encoded by spr1057 gene in Streptococcus pneumoniae, a likely homolog of Escherichia coli protein YjjG. Bacterial cells are constantly exposed to innumerable toxic substances, either in their external environment or by by-products of their own metabolism. For these reasons, the bacterial cells evolved several mechanisms to cope with this challenge. These mechanisms are represented by: blocking the uptake, export by specific transporters as well as specific inactivation of these substance by enzymes. A particular group of these toxic substances are noncanonica nucleotides, which can directly inhibit bacterial cell DNA replication or can result in increased mutation rate. Enzymes recognizing these modified derivatives are known as "house-cleaning" nucleotide phsphateses, which can inactivate the potentially mutagenic nucleotides and prevent their incorporation into DNA and RNA. Some of the "house- cleaning" enzymes belong to a group of haloacid dehalogenase enzymes (haloacid dehalogenase-like hydrolase superfamily), which are found in many bacterial species. This thesis is focused on the function of hypothetical protein Spr1057 of Streptococcus pneumoniae with an unknown function. Sequence comparison revealed that Spr1057 has a significant...

To overcome stresses such as osmotic shock and pathogen attack, internal signals activate plant defense responses. The F8A24.12 gene from Arabidopsis thaliana encodes the serine/threonine protein kinase with the highest similarity at the structural and functional level to a salt-stress induced gene, Esi47 , from the salt tolerant wheatgrass Lophopyrum elongatum . Both genes have a small upstream open reading frame (suORF) in their 5 ' untranslated region (5 ' UTR). This element was previously shown to have a regulatory function in the Esi47 gene. Northern blot analysis revealed that under salt stress both genes have an increased level of transcription. Transgenic Arabidopsis plants were produced carrying reporter gene encoding the enzyme Ý-glucuronidase (GUS) under the control of the F8A24.12 promoter and 5 ' UTR. GUS histochemical assays revealed that F8A24.12 gene expression occurs throughout the life of the plant, particularly in the root elongation zone, in the vascular tissues of roots and leaves, in the flower and in the petal and sepal abscission zone. GUS fluorometric assays demonstrated that the quantity of GUS protein is induced by methyl jasmonate (MeJA) treatment after 12, 24 and 36 hrs and by salt stress after 24 hrs treatment. These data suggest that the F8A24.12 gene is part of the jasmonic acid signaling pathway. This phytohormone is related to the plant growth and plant defense responses. GUS fluorometric assays were also performed on transgenic Arabidopsis carrying an altered suORF promoter-GUS fusion. Mutation of the suORF obliterated the MeJA and salt responsiveness of the F8A24.12 gene promoter plus 5 ' UTR transgene.

Indiana University-Purdue University Indianapolis (IUPUI)
RNA Polymerase II (RNAPII) is responsible for transcription of messenger RNA (mRNA) and many small non-coding RNAs. Progression through the RNAPII transcription cycle is orchestrated by combinatorial posttranslational modifications of the C-terminal domain (CTD) of the largest subunit of RNAPII, Rpb1, consisting of the repetitive sequence (Y1S2P3T4S5P6S7)n. Disruptions of proteins that control CTD phosphorylation, including the phosphatase Rtr1, cause defects in gene expression and transcription termination. There are two described RNAPII termination mechanisms. Most mRNAs are terminated by the polyadenylation-dependent cleavage and polyadenylation complex. Most short noncoding RNAs are terminated by the Nrd1 complex. Nrd1-dependent termination is coupled to RNA 3' end processing and/or degradation by Rrp6, a nuclear specific subunit of the exosome. The Rrp6-containing form a 3'-5' exonuclease complex that regulates diverse aspects of nuclear RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs). It remains unclear whether Rrp6 is directly involved in termination. We discovered that deletion of RRP6 promotes extension of multiple Nrd1-dependent transcripts resulting from improperly processed 3' RNA ends and faulty transcript termination at specific target genes. Defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of Nrd1 depletion from the nucleus. Our data indicate Rrp6 acts with Nrd1 globally to promote transcription termination in addition to RNA processing and/or degradation. Furthermore, we found that deletion of the CTD phosphatase Rtr1 shortens the distance of transcription before Nrd1-dependent termination of specific regulatory antisense transcripts (ASTs), increases Nrd1 occupancy at these sites, and increases the interaction between Nrd1 and RNAPII. The RTR1/RRP6 double deletion phenocopies an RRP6 deletion, indicating that the regulation of ASTs by Rtr1 requires Rrp6 activity and the Nrd1 termination pathway.

Regulated exocytosis enables numerous critical cellular functions such as wound healing, fertilisation, and neurotransmission. In most eukaryotic cells, proteins and other compounds that need to be secreted into the extracellular space are sorted into secretory vesicles, which then translocate to the plasma membrane where they dock and undergo priming reactions. Upon elevation of intracellular calcium levels (caused by a physiological stimulus), release-ready secretory vesicles fuse with the plasma membrane and release their contents. The late, calcium regulated steps of exocytosis (including docking, priming, and fusion) have been studied using a variety of model systems; in particular, studies utilising sea urchin eggs, cell surface complexes (large sheets of plasma membrane with endogenous, docked vesicles) and isolated cortical vesicles have provided numerous original insights into fundamental, conserved molecular mechanisms (Reviewed in Chapter One, and methods pertaining to the use of the model are detailed in Chapter Two). In Chapter Three, using intact eggs and cell surface complexes, I test the hypothesis that the fully primed, release-ready state is one of stable hemifusion (i.e. with the contacting monolayers of the vesicle and plasma membrane having already merged). Studies utilising fluorescent dyes with well-defined membrane properties and membrane permeable cationic amphiphiles capable of disrupting hemifusion intermediates indicate that, contrary to current opinion, the release-ready state is dynamic, and capable of transiently transitioning between hemifusion and close bilayer apposition. The phosphorylation of proteins and lipids has been implicated in modulating all stages of regulated exocytosis, and in Chapter Four I use cortical vesicles and minimised docking and membrane fusion assays to screen a range of well characterised small molecule modulators of phosphorylation. Data from the screen implicate phosphatases and casein kinase 2 in modulating the calcium sensitivity of fusion, and sphingosine kinase in modulating the ability to fuse. As detailed in Chapter Five, combined molecular and functional analysis of cell surface complexes and cortical vesicles with altered membrane sphingolipid levels confirmed that a critical balance of sphingolipids and sphingolipid metabolites is needed to maintain efficient docking, calcium sensitivity, and capacity to fuse.