The structure and mechanism of the Protein Tyrosine Phosphatase-like Phytases (PTPLPs) from Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) were investigated using a combination of enzyme kinetics, site-directed mutagenesis, and X-ray crystallography. I show that PTPLPs use a classical protein tyrosine phosphatase catalytic mechanism and adopt a core PTP fold. Several unique structural features of PTPLPs confer specificity for inositol phosphates. The effect of ionic strength and oxidation on the kinetics and structure of PTPLPs was investigated. The structural consequences of reversible and irreversible oxidation on PTPLPs and PTPs are compared and discussed. We determine the structural basis of substrate specificity in PTPLPs and propose a novel reaction mechanism for the hydrolysis of inositol polyphosphates by PTPLPs. Finally, the structure and function of a unique tandemly repeated phytase has been determined. We show that the active sites of the tandem repeat possess significantly different specificities for inositol polyphosphate. / xix, 148 leaves : ill. (some col.) ; 29 cm

A novel bacterial protein tyrosine phosphatase (PTP)-like enzyme has recently been isolated that has a PTP-like active site and fold and the ability to dephosphorylate myo-inositol hexakisphosphate. In order to expand our knowledge of this novel class of enzyme, four new representative genes were cloned from 3 different anaerobic bacteria related to clostridia and the recombinant gene products were examined. A combination of site-directed mutagenesis, kinetic, and high-performance ion-pair chromatography studies were used to elucidate the mechanism of hydrolysis, substrate specificity, and pathways of Ins P6 dephosphorylation. The data indicate that these enzymes follow a classical PTP mechanism of hydrolysis and have a general specificity for polyphosphorylated myo-inositol substrates. These enzymes dephosphorylate Ins P6 in a distributive manner, and have the most highly ordered pathways of sequential dephosphorylation of InsP6 characterized to date. Bioinformatic analyses have indicated homologues that are involved in the regulation of cellular function. / x, 138 leaves ; 29 cm.

Programmed cell death (PCD) is an active process by which organisms coordinate the controlled destruction of cells. In tomato, the protein kinase Adi3 (AvrPto-dependent Pto-interacting kinase 3), acts as a negative regulator of PCD and shares important functional homologies with the mammalian anti-apoptotic AGC kinase PBK/Akt. Adi3 was originally identified as an interactor of the complex formed by the tomato resistance protein Pto and the Pseudomonas syringae pv. tomato (Pst) effector protein AvrPto. The complex formed by AvrPto and Pto causes a resistance response characterized by a rapid form of PCD that limits the spread of Pst and prevents the onset of the tomato bacterial speck disease. In an effort to characterize the mechanisms by which Adi3 regulates PCD, we identified Adi3 interacting partners in a Y2H screen. Here, I describe the interaction of Adi3 with two interacting partners identified: the Sucrose Non-fermenting (SNF1) kinase complex (SnRK) which is a eukaryotic master regulator of energy homeostasis and the E3 RING Ubiquitin ligase AdBiL. Using a combination of in vitro and in vivo approaches I found that AdBiL is an active ubiquitin ligase that ubiquitinates Adi3. Interestingly, Adi3 was found to be degraded in a proteasome-dependent manner suggesting ubiquitination could play a role in its degradation. On the other hand, Adi3 was found to inhibit the SnRK complex by directly interacting with its catalytic subunit as well as by phosphorylating the regulatory subunit SlGal83 at Ser26. SlGal83 is phosphorylated at multiple sites in vivo, and this phosphorylation state, as well as its intracellular localization was found to depend on a myristoylation signal present at its N-terminus. Phosphorylation at Ser26 by Adi3 was found to alter the localization of this subunit in a myristoylation-dependent manner. The interactions studied in this dissertation provide additional evidence on the functional homologies shared by Adi3 and PKB. In addition, the regulatory control of SnRK activity and cellular localization offers a novel connection between pathways involved in energy homeostasis and pathogen-mediated PCD.

PEPC [PEP(phosphoenolpyruvate) carboxylase] is an essential and tightly controlled enzyme located at the core of plant C-metabolism. It fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and N-assimilation. In plants, a small multigene family encodes several closely related plant-type PEPC (PTPC) isozymes along with a distantly related bacterial-type PEPC (BTPC) isozyme. The PTPCs are well studied ~110-kDa subunits that typically exist as a homotetramer (Class-1 PEPC). By contrast, little is known about the larger ~118-kDa BTPC isozyme except that it occurs in developing castor (Ricinus communis) endosperm in tight association with PTPC subunits as a ~900-kDa hetero-octameric complex (Class-2 PEPC) that is greatly desensitized to metabolic effectors compared to Class-1 PEPC. This thesis elucidates the physiological purpose of the BTPC subunits by examining their structure/function relationship within Class-2 PEPC and identifying mechanisms of post-translational control. Recombinant expression and purification of the castor bean BTPC revealed unusual physical and kinetic properties including a remarkable insensitivity to metabolic effectors and a dependence upon PTPC subunits for structural stability. The first purification of a non-proteolyzed plant Class-2 PEPC complex was performed, and the kinetic analysis determined that the BTPC and PTPC subunits have complimentary catalytic properties. The BTPC subunits’ high Km(PEP) and desensitization to metabolic effectors may function as a metabolic overflow mechanism for sustaining flux from PEP to malate when PTPC subunits become feedback inhibited. An anti-PTPC co-immunopurification strategy was utilized to highly enrich non-proteolyzed BTPC from developing castor endosperm for downstream immunological and mass spectrometric analysis. BTPC was in vivo phosphorylated at multiple novel sites, identified by mass spectrometry as Thr4 or 5, Ser425 and Ser451. Phosphosite-specific antibodies towards Ser425 and Ser451 confirmed the existence of these sites in vivo and comparisons of Ser425 phosphorylation patterns established that the castor BTPC and PTPC phosphorytation sites are regulated independently. Phosphomimetic mutants of Ser425 caused BTPC inhibition by increasing its Km(PEP) and sensitivity to feedback inhibition. These results establish a novel mechanism of PEPC control whose implications within plant carbon metabolism are discussed. / Thesis (Ph.D, Biology) -- Queen's University, 2011-09-04 16:46:22.024

The effects of temperature on a 58-kDa phosphoprotein (PP58) have been examined in cell-free extracts of two alfalfa (Medicago sativa L.) cultivars, Apica and Trek. In the extracts prepared without the use of Triton X-100, PP58 is present in a 12,000 x g (P12), 28,000 x g (P28) and 100,000 x g (P100) pellets but is enriched in the P28 fraction. In these fractions PP58 is substantially and equally phosphorylated at both 4° and 24°C. When extracts are prepared in the presence Triton X-100, PP58 is present in the 28,000 x g supernatant (TXS fraction) is extensively dephosphorylated at 24°C but highly phosphorylated at 4°C. The phosphorylation of this protein increased sharply as temperature declined below 12°C, and was 15 times greater at 0° than at 24°C. The phosphorylation level doubled between 12° and 8°C and again between 8° and 4°C. Thus temperature effect is not mediated by Q10 effect. Interestingly, temperature-response curve of PP58 phosphorylation is similar to that of the reported cold-induced calcium influx (Plant Cell 7: 321-331). Labeling reactions carried out in the presence of [gamma-35S]thioATP indicated that low temperature inhibited the dephosphorylation reaction. These results were not mimicked at room temperature by the protein phosphatase 1 and 2A inhibitor okadaic acid. In reactions performed at 4°C, addition of calcium caused a 2-fold increase in the phosphorylation of PP58. A decrease in phosphorylation was observed when equimolar amounts of EGTA were added in the presence of MgCl 2 or MnCl2, but not in the presence of CaCl2, suggesting that this protein is phosphorylated by a calcium-dependent protein kinase. These results are consistent with the suggestion that PP58 and its putative kinase are membrane-localized whereas the putative PP58 phosphatase is a loosely-associated membrane peripheral protein lost to the supernatant during fractionation. We suggest that PP58 could be involved in low te

La voie de signalisation Notch contient quatre récepteurs transmembranaires qui sont activés de façon ligand dépendant. Les contacts cellulaires permettent l'activation des récepteurs et la relâche entre autres du fragment actif Notch 1 clivé (NIC1). Ensuite, il transloque au noyau pour s'associer à son co-activateur, CSL afin de permettre la formation d'un complexe transcriptionnel actif. La signalisation Notch est régulée à divers niveaux cellulaires. La voie Notch est très engagée durant le développement du pancréas. De plus, différents modèles ont été générés afin de mieux comprendre l'implication de la voie Notch dans le phénomène de la prolifération des cellules pancréatiques Ainsi, la voie Notch est active dans le pancréas immature et est réprimée dans le tissu adulte. La signalisation Notch est très importante pendant la carcinogenèse pancréatique. Il a été démontré que l'inhibition de la voie Notch induit une répression de la croissance cellulaire et bloque la tumorigenèse pancréatique. Une autre voie de signalisation très impliquée dans le cancer du pancréas est la voie KRas qui est mutée dans 90% des adénocarcinomes. Une coopération entre les voies Notch et KRas a été reportée dans différents types de carcinogenèse. De plus, il a été suggéré que la collaboration Notch et KRas passe spécifiquement par la cascade Mek/Erk en aval de KRas. De plus, l'inhibition de la voie Mek/Erk mène à l'inhibition de l'expression d'Hes1, une cible de la voie Notch. Ainsi, nous avions supposé que la cascade KRas/Raf/Mek/Erk favorise la transcription induite par la voie de signalisation Notch. Pour y répondre, il était donc important de déterminer l'impact de la voie Mek/Erk sur l'expression de Notch 1 clivé (NIC1) et de clarifier l'effet de cette voie sur son activité transcriptionnelle. Nous avons observé que la suractivation de la voie Mek/Erk induit un retard de migration de la protéine NIC1 qui est complètement aboli par l'inhibition de la voie Mek/Erk. Des études plus approfondies ont révélé que ces formes de plus haut poids moléculaires étaient des formes phosphorylées de NIC 1 qui pouvaient être associées directement à l'action des Erk 1. Nous avons découvert que ces modifications permettent de réguler l'activité transcriptionnelle de Notch. Ainsi, la phosphorylation de NIC1 induite par la voie Mek/Erk est associée à une augmentation de l'activité transcriptionnelle dépendante de Notch qui mène à l'expression du gène cible, Hes1. Il semble aussi que le domaine PEST de NIC 1 est essentiel afin de permettre les phosphorylations Erk-dépendantes. D'autres effets spécifiques de la voie Mek/Erk sur Notch restent à être découverts. Enfin, le potentiel de cette collaboration entre les voies Notch et KRas/Raf/Mek/Erk pourrait être impliquée dans divers processus cellulaires.

No description available.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 115-129). / Microfiche. / ix, 129 leaves, bound ill. 29 cm

Research Doctorate - Doctor of Philosopy (PhD) / Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated acutely by a combination of phosphorylation of three key serine (Ser) residues (Ser19, Ser31 and Ser40), and feedback inhibition by the catecholamines. Phosphorylation of Ser40 directly increases TH activity by relieving feedback inhibition of the enzyme. The phosphorylation of Ser19 or Ser31 can potentiate the phosphorylation of Ser40 in a process known as hierarchical phosphorylation. The 2 major human TH isoforms, hTH1 and hTH2, are differentially regulated by hierarchical phosphorylation in vitro. In this study, the human neuroblastoma SH-SY5Y cell line has been transfected with hTH1 and hTH2, and it has been demonstrated that phosphorylation of Ser31 potentiates the phosphorylation of Ser40 in hTH1. Phosphorylation of the equivalent Ser31 residue in hTH2 was not detectable, and thus this enzyme is not subject to Ser31-mediated hierarchical phosphorylation of Ser40 in situ. This is the first study to demonstrate that hTH1 and hTH2 are differentially regulated in situ. In addition, we have examined the nature of feedback inhibition of TH by the catecholamines. In addition to the high affinity, non-dissociable dopamine binding that is relieved by Ser40 phosphorylation, we have identified a second low affinity, readily dissociable binding site which regulates TH activity both in vitro and in situ regardless of the phosphorylation state of the enzyme. This low affinity binding site responds to changes in cytosolic catecholamine levels in situ in order to regulate TH activity. This work has contributed to our understanding of the complex nature of the regulation of TH activity.

Macrophages serve key roles in host defence by initiating inflammatory responses to infection and/or injury. They contribute to innate immunity by secreting a range of pro-inflammatory cytokines (e.g. TNF and IL-6) upon activation as well as by phagocytosing pathogens and dead cells, which is necessary for the resolution of inflammation and effective wound repair. Macrophages also contribute to adaptive immunity by functioning as antigen presenting cells.Colony stimulating factor 1 (CSF-1) is the major growth factor governing the differentiation, proliferation and survival of macrophages. Although not as well appreciated, CSF-1 also regulates some of the immune functions of macrophages, such as cytokine secretion and phagocytosis. However, the mechanisms by which CSF-1 governs the immune functions of macrophages are poorly understood. Cytokine secretion, phagocytosis and antigen presentation involve various vesicle trafficking and membrane fusion events, processes in which SNARE proteins play vital roles. Thus, the hypothesis tested in this thesis was that CSF-1 modulates the immune functions of macrophages by regulating the expression and/or activity of SNARE proteins that regulate endocytic and exocytic processes.In this study, the endosomal SNARE protein syntaxin 7 was identified, via microarray analysis, as a CSF-1 inducible gene in primary mouse macrophages. Syntaxin 7 has previously been detected in phagosomal membranes in macrophages. Furthermore, syntaxin 7 has recently been implicated in the secretion of cytokines (e.g. TNF) from macrophages by forming a novel complex with syntaxin 6, Vti1b and VAMP3.