No description available.

No description available.

No description available.

No description available.

The 55K product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation. Both biochemical and genetic approaches have been used to show that this 496-residue (496R) protein of adenovirus (Ad5) is phosphorylated at both serine and threonine residues at sites near the carboxy terminus within sequences characteristic of a casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased virus replication and reduced the efficiency of transformation of primary baby rat kidney cells, suggesting that phosphorylation at these sites is of importance in the regulation of 55K biological activity. / Thesis / Master of Science (MS)

For many years, it has been understood that protein phosphorylation-dephosphorylation constitutes one of the most ubiquitous mechanisms for controlling the functional properties of proteins. Although originally believed to be a eukaryotic phenomenon, protein phosphorylation is now known to occur in all three domains of life <i>Eukarya, Bacteria,</i> and Archaea</i>. Very little is known, however, concerning the origins and evolution of protein phosphorylation-dephosphorylation. Knowledge of the structure and properties of the protein kinases resident in the members of the <i>Archaea</i> represents a key piece of this puzzle. The extreme acidothermophilic archaeon, <i>Sulfolobus solfataricus</i>, exhibits a membrane-associated protein kinase activity. Solubilization of the kinase activity requires the presence of detergent such as Triton X-100 or octyl glucoside, indicating its activity reside in an integral membrane protein. This protein kinase utilizes purine nucleotides as phosphoryl donors <i>in vitro</i> with a requirement for a divalent metal ion cofactor, favoring Mn⁺². A preference for NTPs over NDPs and for adenyl nucleotides over the analogous guanyl nucleotides was observed. The enzyme appears to be a glycoprotein that displays catalytic activity on SDS-PAGE corresponding to a molecular mass of ≈67 kDa, as well as an apparent molecular mass of –125 kDa on a gel filtration column. Challenged with several exogenous substrates revealed the protein kinase to be relatively selective. Only casein, reduced carboxyamidomethylated and maleylated lysozyme (RCM lysozyme), histone H4 proved, and a peptide modeled after myosin light chains (KKRAARATSNVFA) were phosphorylated to appreciable levels <i>in vitro</i>. All of the aforementioned substrates were phosphorylated on threonine, while histone H4 was phosphorylated on serine as well. When the phosphoacceptor threonine in the MLC peptide was substituted with serine an appreciable decrease in phosphorylation was noted. The protein kinase underwent autophosphorylation on threonine and was relatively insensitive to several known "eukaryotic" protein kinase inhibitors. Primary sequence motifs based on known conserved subdomains of eukaryotic protein kinases were used to search the genome of <i>S. solfataricus</i> for eukaryotic-like protein kinase sequences. Six hypothetical proteins were identified from <i>S. solfataricus</i> whose primary sequence exhibited noticeable similarities to eukaryotic protein kinases. The hypothetical protein encoded by <i>ORF sso0197</i> contained 7 putative subdomains, <i>ORFs sso0433, sso2291, sso2387</i>, and <i>sso3207</i> contained 8 putative subdomains, and <i>ORF sso3182</i>, contained 9 putative subdomains of the 12 characteristically conserved subdomains found within eukaryotic protein kinases. <i>ORF sso2387</i> was cloned and expressed in <i>Escherichia coli</i>. The expressed protein, SsPK2, was solubilized from inclusion bodies using 5 M urea. SsPK2 was able to phosphorylate casein, BSA, RCM lysozyme, and mixed histones <i>in vitro</i>. Phosphoamino acid analysis of casein, BSA, and mixed histones revealed that they were all phosphorylated on serine. SsPK2 underwent autophosphorylation on serine at elevated temperature using both purine nucleotide triphosphates as phosphoryl donors in vitro, but exhibited a noticeable preference for ATP. Autophosphorylate of SsPK2 also occurred at elevated temperature using a variety of divalent metals cofactors in order of Mn⁺² > Mg⁺² >> Ca²⁺ ≈ Zn⁺². Polycations such as polyLys stimulated the phosphorylation of exogenous substrates while polyanions such as poly(Glu:Tyr) were shown to inhibit the phosphorylation of exogenous substrates. Of the "eukaryotic" protein kinases inhibitors tested, only tamoxifen had any noticeable effect of the catalytic activity of SsPK2 towards itself and exogenous substrates. A truncated form of SsPK2 containing the perceived catalytic domain also exhibited protein kinase activity towards itself and exogenous substrates. The observed protein kinase activity for SsPK2trunk was similar to that observed for SsPK2. Proteins from the membrane fraction of <i>S. solfataricus</i> subject to phosphorylation <i>in vitro</i> on serine or threonine residues were identified using MALDI-MS / peptide fingerprinting techniques. Nine phosphoproteins were assigned a tentative identification using the ProFound protein search engine from Rockefeller University. The identity of two of nine phosphoproteins, a translational endoplasmic reticulum ATPase and an ≈ 42 kDa hypothetical protein, were determined with a relatively high degree of confidence. Collectively the results suggested MALDI-MS peptide mapping coupled with [³²P] labeling <i>in vivo</i> will have a tremendous potential for mapping out a major portion of the phosphoproteome of <i>S. solfataricus</i>. / Ph. D.

La liaison de l'insuline à son récepteur de surface entraîne une autophosphorylation rapide de la sous-unité β du récepteur de l'insuline (RI), l'initiation de cascades de signalisation et l'internalisation rapide de complexes actifs dans l'appareil endosomal. L'autophosphorylation s'effectue sur trois tyrosines situées dans la boucle d'activation (Yl 146, Y1150, Y1151), deux tyrosines situées dans le domaine C-terminal (Y1316, Y1322) et d'une autre dans le domaine juxta-membranaire (Y960). Ces sites de phosphorylation ont été précédemment identifiés par des essais d'autophosphorylation effectués in vitro sur des fractions membranaires solubilisées. Dans le but d'identifier les sites tyrosines phosphorylés du RI internalisé in vivo, nous avons préparé des fractions Golgi/Endosomes (G/E) à partir de foie de rat suivant l'injection d'une dose d'insuline. Le RI a été immunoprécipité et analysé par spectrométrie de masse. Plusieurs sites de phosphorylation ont été identifiés et seules les formes mono phosphorylé Y1146 et Y1150 de la boucle d'activation ont été observées. Nous avons également identifié de façon non ambiguë plusieurs protéines associés au RI internalisé dont notamment Grb7 (growth factor receptor-bound protein 7), Grbl4 et ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase). Nous avons confirmé, par des immunoprécipitations croisées dans les fractions G/E, l'association entre le RI et ATIC, une protéine impliquée dans les deux dernières étapes de la voie de biosynthèse de novo des purines. Les résultats obtenus suggèrent la présence d'un nouveau mécanisme par lequel ATIC, une protéine relativement abondante dans le cytosol et ayant pour substrat naturel AICAR, un insulino-mimétique, pourrait être un régulateur important de la réponse insulinique in vivo.

L'incidence de maladies neurodegeneratives et systémiques liées au vieillissement, tels la maladie d'Alzheimer (MA) et le diabète, augmente rapidement. Plusieurs études rapportent que les patients souffrant de diabète ont entre 50 et 75 % plus de risque de développer la MA que les gens sains du même âge. La protéine tau hyperphosphorylée est une des composantes majeures des enchevêtrements neurofibrillaires, une composante neuropathologique classique de la MA. L'étude présente examine les modifications de tau dans deux modèles de souris transgéniques développant un diabète de type 2. La phosphorylation de tau est augmentée au niveau de l'hippocampe de ces souris. Le diabète de type 2 provoque aussi la dérégulation de quelques kinases et phosphatases de tau. Cette dérégulation serait résultante de la résistance à l'insuline et l'hypothermie. Le diabète de type 2 provoquerait donc l'accélération de la cascade menant à développer la maladie d'Alzheimer.

HPr fait partie du système de transport phosphoénolpyruvate: sucre phosphotransferase (PTS). HPr peut être phosphorylée sur Hisis, par l'enzyme I (El) du PTS, et sur Sér46, par la HPr(Ser) kinase/phosphorylase (HprK/P). Finalement, HPr peut être phosphorylée sur les deux résidus, ce qui génère HPr(Ser-P)(His~P). L'objectif principal de l'étude présentée dans cette thèse était de déterminer par quelle(s) voie(s) HPr(Ser-P)(His~P) est synthétisée chez les streptocoques. Théoriquement, HPr(Ser-P)(His~P) peut être synthétisée via la phosphorylation de HPr(Ser-P) par El et/ou de HPr(His-P) par HprK/P. Nous avons étudié la cinétique de ces voies de synthèse chez Streptococcus salivarius. Les résultats de l'étude avec El ont montré que (i) la kcat/Km pour HPr(Ser-P) était ~5 OOOx plus faible que pour HPr à pH 7,9 et 37°C, (ii) aucun intermédiaire glycolytique ne stimulait la synthèse de HPr(Ser-P)(His~P), (iii) la synthèse de HPr(Ser-P)(His~P) était ~8x plus efficace à pH acide, des conditions retrouvées dans le cytoplasme des streptocoques en croissance, et (iv) la synthèse du HPr(Ser-P)(His~P) de Bacillus subtilis par El de cet organisme était aussi stimulée à pH acide. Les résultats suggèrent que la synthèse de HPr(Ser-P)(His~P) chez les streptocoques résulterait des concentrations élevées en HPr(Ser-P) et de la stimulation de la réaction à pH acide. L'absence de HPr(Ser-P)(His~P) chez B. subtilis cultivée en présence de glucose s'expliquerait en partie par le fait que cette bactérie maintient son pH intracellulaire constamment au-dessus de la neutralité. Les résultats de l'étude avec HprK/P ont montré que les kQJKm pour les HPr(H15D) et HPr(H15E) étaient ~13x plus faibles que pour HPr à pH 7,4 et 37°C. Dans des conditions où HPr(His~P) était stable (pH 8,6 et 15°C), les kcJKm pour HPr(H15D) et HPr(His~P) Ill étaient respectivement neuf et 26 fois plus faibles que pour HPr. Finalement, la phosphorylation de HPr(H15D) n'était que marginalement stimulée à pH acide et le FBP ne stimulait pas la synthèse de HPr(Ser-P)(His~P). Les résultats suggèrent que (i) l'inefficacité de la phosphorylation de HPr(His~P) résulterait de la présence de la charge négative en position 15 et d'autres éléments structuraux et (ii) la contribution de HprK/P à la synthèse de HPr(Ser-P)(His~P) chez les streptocoques serait marginale.

Synaptophysin (Syp) is a major integral synaptic vesicle (SV) protein; there are 31 copies of Syp per vesicle, which totals up to 10% of the total SV protein content. Despite being the major SV protein, little is known about the interaction partners of Syp and as a result there has been no clear role attributed to it. One key feature of Syp is that its cytoplasmic C-terminus contains 10 pentapeptide repeats, nine of which are initiated by a tyrosine residue. Syp is the major tyrosine phospho-protein on SVs. The kinase thought to phosphorylate Syp in vivo is the ubiquitously expressed non-receptor kinase C-Src. There are two splice variants of C-Src, N1- and N2-Src, which are only expressed in neuronal tissues. Although the 3 Srcs are structurally similar, they differ by a small insert of amino acids into their SH3 domains (the N-Src loop). Examination of the amino acid sequence of the cytosolic C-terminus of Syp revealed a putative type one SH3 domain interaction motif. A screen using SH3 domains of synaptic proteins as bait in GST-pull downs from nerve terminal lysate allowed an inventory of potential interaction partners of Syp to be created. Reciprocal experiments using the C-terminal of Syp as bait confirmed many of these interactions. Single point mutations of the SH3 interaction motif on Syp highlighted that syndapin and C-Src bound to Syp via this motif. These binding mutants were inserted in Syp superecliptic synaptophluorin (SypHy) to determine the functional consequences of these interactions. These mutants did not affect the trafficking of Syp when expressed in cortical neurons derived from Syp knockout mice. However, the SH3 interaction motif was fundamental for the retrieval of VAMP (vesicle associated membrane protein) when expressed in Syp knockout cultures. Importantly, this role is not mediated through a direct interaction with VAMP with the SH3 interaction motif implicating either syndapin, C-Src or both in Syp-dependent VAMP retrieval. The 3 different Srcs had different methods of interaction with Syp, and in vitro protein kinase assays the ability of the three Src splice variants to phosphorylate Syp was assessed. Key differences in both speed and efficiency of Syp phosphorylation was observed for the different Src splice variants. Mutagenesis of either all 9 tyrosine residues, only previously identified sites resulted in changes in Syp interactions in GST-pull down assays from nerve terminal lysates. To investigate the role of Syp phosphorylation in the SV lifecycle, the tyrosine pentapeptide repeats were truncated from the C-terminal of Syp in both a mCerulean tagged Syp and SypHy. The experiments showed that these potential tyrosine phosphorylation sites were not involved in the trafficking of Syp but key in the retrieval of VAMP from the plasma membrane during the SV lifecycle. I have indentified an SH3 interaction motif on the C-terminal of Syp that is critical in forming a complex of proteins that are responsible for the retrieval of VAMP during the SV lifecycle. Further experiments have shown that this key interaction is potentially phosphorylation dependent. My preliminary mass spectrometry analysis has provided a catalogue of proteins that can potentially interact with Syp, identifying proteins that may bind to either the Syp C-terminus SH3 interaction motif or to other regions in a phosphorylation dependent manner. This has provided a list of potential candidate proteins for the VAMP retrieval complex.