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Characterization of Phosphorylation Sites on the Glutamate Receptor 4 Subunit of the AMPA Receptors

Carvalho, Ana Luísa; Kameyama, Kimihiko; Huganir, Richard L.
Fonte: Society for Neuroscience Publicador: Society for Neuroscience
Tipo: Artigo de Revista Científica
ENG
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36.41%
Recent studies have suggested that protein phosphorylation of glutamate receptors may play an important role in synaptic transmission. Specifically, the phosphorylation of AMPA receptors has been implicated in cellular models of synaptic plasticity. The phosphorylation of the glutamate receptor 1 (GluR1) subunit of AMPA receptors by protein kinase A (PKA), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized extensively. Phosphorylation of this subunit occurs exclusively on the intracellular C-terminal domain. However, the GluR1 subunit C terminus shows low homology to the other AMPA receptor subunits. In this paper we characterized the phosphorylation of AMPA receptor subunit GluR4, using site-specific mutagenesis and biochemical techniques. We found that GluR4 is phosphorylated on serine 842 within the C-terminal domain in vitro and in vivo. Serine 842 is phosphorylated by PKA, PKC, and CaMKII in vitro and is phosphorylated in transfected cells by PKA. Two-dimensional phosphopeptide analysis indicates that serine 842 is the major phosphorylation site on GluR4. In addition, we identified threonine 830 as a potential PKC phosphorylation site. These results suggest that GluR4, which is the most rapidly desensitizing AMPA receptor subunit...

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

Freire, Paula Paccielli; Alves, Carlos Augusto Barnabe; Deus, Adriana Fernandes De; Leopoldo, Ana Paula Lima; Leopoldo, André Soares; Silva, Danielle Cristina Tomaz Da; Tomasi, Loreta Casquel De; Campos, Dijon Henrique Salomé; Cicogna, Antonio Carlos
Fonte: Sociedade Brasileira de Cardiologia (SBC) Publicador: Sociedade Brasileira de Cardiologia (SBC)
Tipo: Artigo de Revista Científica Formato: 0-0
ENG
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Fundamento:A ativação do sistema beta-adrenérgico promove a estimulação da proteína G, que, via adenosina monofosfato cíclico (AMPc), altera a estrutura da proteina quinase A (PKA) e acarreta a fosforilação da fosfolambam (PLB). Essa proteína participa do sistema envolvido no controle de cálcio intracelular, em células musculares, sendo a principal reguladora da atividade da bomba de cálcio do retículo sarcoplasmático. Na obesidade ocorre ativação do sistema beta-adrenérgico por influência do aumento da leptina, acarretando, consequentemente, maior fosforilação da fosfolambam miocárdica, via AMPc-PKA.Objetivo:Investigar, na obesidade, o envolvimento das proteínas que regulam o grau de fosforilação do PLB decorrente da ativação beta-adrenérgica. A hipótese do estudo é que há desequilíbrio entre a fosforilação e a desfosforilação da fosfolambam, com predomínio da fosforilação da proteína.Métodos:Ratos Wistar machos foram randomizados e distribuídos em dois grupos: controle (n = 14), alimentado com dieta normocalórica, e obeso (n = 13), com um ciclo de quatro dietas hiperlipídicas insaturadas. A obesidade foi determinada pelo índice de adiposidade, e as expressões proteicas de fosfatase 1 (PP-1)...

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

Freire, Paula Paccielli; Alves, Carlos Augusto Barnabe; Deus, Adriana Fernandes De; Leopoldo, Ana Paula Lima; Leopoldo, André Soares; Silva, Danielle Cristina Tomaz Da; Tomasi, Loreta Casquel De; Campos, Dijon Henrique Salomé; Cicogna, Antonio Carlos
Fonte: Sociedade Brasileira de Cardiologia - SBC Publicador: Sociedade Brasileira de Cardiologia - SBC
Tipo: Artigo de Revista Científica Formato: 41-50
ENG
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36.38%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Background:The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. Objective:To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Methods:Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index...

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

Freire,Paula Paccielli; Alves,Carlos Augusto Barnabe; Deus,Adriana Fernandes de; Leopoldo,Ana Paula Lima; Leopoldo,André Soares; Silva,Danielle Cristina Tomaz da; Tomasi,Loreta Casquel de; Campos,Dijon Henrique Salomé; Cicogna,Antonio Carlos
Fonte: Sociedade Brasileira de Cardiologia - SBC Publicador: Sociedade Brasileira de Cardiologia - SBC
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/07/2014 EN
Relevância na Pesquisa
36.38%
Background: The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. Objective: To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Methods: Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot. Results: Obesity caused glucose intolerance...

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/1997 EN
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The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores...

Régulation dynamique de l’activité du récepteur des estrogènes beta (ERβ) par la phosphorylation,l’ubiquitination et la sumoylation

Picard, Nathalie
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
FR
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36.4%
Les estrogènes jouent un rôle primordial dans le développement et le fonctionnement des tissus reproducteurs par leurs interactions avec les récepteurs des estrogènes ERα et ERβ. Ces récepteurs nucléaires agissent comme facteurs de transcription et contrôlent l’expression des gènes de façon hormono-dépendante et indépendante grâce à leurs deux domaines d’activation (AF-1 et AF-2). Une dérégulation de leur activité transcriptionnelle est souvent à l’origine de pathologies telles que le cancer du sein, de l’endomètre et des ovaires. Alors que ERα est utilisé comme facteur pronostic pour l’utilisation d’agents thérapeutiques, l’importance de la valeur clinique de ERβ est encore controversée. Toutefois, des évidences récentes lui associent un pouvoir anti-tumorigénique en démontrant que sa présence favorise l’inhibition de la progression de ces cancers ainsi que l’efficacité des traitements. En combinaisons avec d’autres études, ces observations démontrent que bien que les deux isoformes partagent une certaine similitude d’action, les ERs sont en mesure d’exercer des fonctions distinctes. Ces différences sont fortement attribuables au faible degré d’homologie observé entre certains domaines structuraux des ERs...

In Vivo Phosphorylation of Serine-451 in the Bacterial-Type Phosphoenolpyruvate Carboxylase from Developing Castor Oil Seeds

Brikis, Carolyne
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado
EN
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36.44%
Class-2 phosphoenolpyruvate carboxylase (PEPC), which includes the enigmatic bacterial-type phosphoenolpyruvate carboxylase (BTPC) subunit, is hypothesized to support carbon flux towards fatty acid synthesis in the developing endosperm of the castor oil seed (COS). COS BTPC was recently discovered to be subject to in vivo multisite phosphorylation at Threonine-4, Serine-425 and Serine-451, which is of major interest due to the importance of phosphorylation as a post-translational protein modification. To further characterize these novel phosphorylation events, a phospho-site specific antibody (anti-(pSer-451)-IgG) was raised against the novel phosphorylation site Ser-451 of developing COS BTPC. The results confirm that the anti-(pSer-451)-IgG is phosphorylation site-specific, and that COS BTPC is in fact phosphorylated in vivo at Ser-451 at a high phosphorylation stoichiometry. The amino acid sequence surrounding Ser-451 is strongly reminiscent of a phosphorylation motif recognized by SnRK1 kinase. Ser-451 exhibited constant phosphorylation throughout COS development and seed depodding but seemed maximal at later developmental stages. Thus, the patterns of phosphorylation do not appear to coincide with developing COS fatty acid or storage protein synthesis. The current study provides insight into the occurrence of phosphorylation on vascular plant BTPC.; NSERC

Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling

Pitson, S.; Xia, P.; Leclercq, T.; Moretti, P.; Zebol, J.; Lynn, H.; Wattenberg, B.; Vadas, M.
Fonte: Rockefeller Univ Press Publicador: Rockefeller Univ Press
Tipo: Artigo de Revista Científica
Publicado em //2005 EN
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Sphingosine kinase (SK) 1 catalyzes the formation of the bioactive lipid sphingosine 1-phosphate, and has been implicated in several biological processes in mammalian cells, including enhanced proliferation, inhibition of apoptosis, and oncogenesis. Human SK (hSK) 1 possesses high instrinsic catalytic activity which can be further increased by a diverse array of cellular agonists. We have shown previously that this activation occurs as a direct consequence of extracellular signal–regulated kinase 1/2–mediated phosphorylation at Ser225, which not only increases catalytic activity, but is also necessary for agonist-induced translocation of hSK1 to the plasma membrane. In this study, we report that the oncogenic effects of overexpressed hSK1 are blocked by mutation of the phosphorylation site despite the phosphorylation-deficient form of the enzyme retaining full instrinsic catalytic activity. This indicates that oncogenic signaling by hSK1 relies on a phosphorylation-dependent function beyond increasing enzyme activity. We demonstrate, through constitutive localization of the phosphorylation-deficient form of hSK1 to the plasma membrane, that hSK1 translocation is the key effect of phosphorylation in oncogenic signaling by this enzyme. Thus...

Kinases and protein phosphorylation as regulators of steroid hormone action

Weigel, N.; Moore, N.
Fonte: Nuclear Receptor Signaling Atlas Publicador: Nuclear Receptor Signaling Atlas
Tipo: Artigo de Revista Científica
Publicado em //2007 EN
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Although the primary signal for the activation of steroid hormone receptors is binding of hormone, there is increasing evidence that the activities of cell signaling pathways and the phosphorylation status of these transcription factors and their coregulators determine the overall response to the hormone. In some cases, enhanced cell signaling is sufficient to cause activation of receptors in medium depleted of steroids. Steroid receptors are targets for multiple kinases. Many of the phosphorylation sites contain Ser/Thr-Pro motifs implicating proline-directed kinases such as the cyclin-dependent kinases and the mitogen-activated kinases (MAPK) in receptor phosphorylation. Although some sites are constitutively phosphorylated, others are phosphorylated in response to hormone. Still others are only phosphorylated in response to specific cell signalling pathways. Phosphorylation of specific sites has been implicated not only in overall transcriptional activity, but also in nuclear localization, protein stability, and DNA binding. The studies of the roles of phosphorylation in coregulator function are more limited, but it is now well established that many of them are highly phosphorylated and that phosphorylation regulates their function. There is good evidence that some of the phosphorylation sites in the receptors and coregulators are targets of multiple signaling pathways. Individual sites have been associated both with functions that enhance the activity of the receptor...

Steroid receptor phosphorylation: a key modulator of multiple receptor functions

Weigel, N.; Moore, N.
Fonte: Endocrine Soc Publicador: Endocrine Soc
Tipo: Artigo de Revista Científica
Publicado em //2007 EN
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Steroid receptors are hormone-activated transcription factors, the expression and activities of which are also highly dependent upon posttranslational modifications including phosphorylation. The remarkable number of phosphorylation sites in these receptors and the wide variety of kinases participating in their phosphorylation facilitate integration between cell-signaling pathways and steroid receptor action. Sites have been identified in all of the functional domains although the sites are predominantly in the amino-terminal portions of the receptors. Regulation of function is receptor specific, site specific, and often dependent upon activation of a specific cell-signaling pathway. This complexity explains, in part, the early difficulties in identifying roles for phosphorylation in receptor function. With increased availability of phosphorylation site-specific antibodies and better means to measure receptor activities, numerous roles for site-specific phosphorylation have been identified including sensitivity of response to hormone, DNA binding, expression, stability, subcellular localization, and protein-protein interactions that determine the level of regulation of specific target genes. This review summarizes current knowledge regarding receptor phosphorylation and regulation of function. As functional assays become more sophisticated...

β integrin tyrosine phosphorylation is a conserved mechanism for regulating talin-induced integrin activation; beta integrin tyrosine phosphorylation is a conserved mechanism for regulating talin-induced integrin activation

Anthis, N.; Haling, J.; Oxley, C.; Memo, M.; Wegener, K.; Lim, C.; Ginsberg, M.; Campbell, I.
Fonte: Amer Soc Biochemistry Molecular Biology Inc Publicador: Amer Soc Biochemistry Molecular Biology Inc
Tipo: Artigo de Revista Científica
Publicado em //2009 EN
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Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the β integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated 15N-labeled β3, β1A, and β7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of β3, β1A, and β7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of β integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis...

Modulation der Insulinsignalübertragung durch Proteinkinase C-katalysierte Phosphorylierung von Serin 318 im Insulinrezeptor-Substrat-1; Modulation of insulin signaling by protein kinase C-induced phosphorylation of insulin receptor substrate-1 at serine 318

Möschel, Klaus
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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Diabetes mellitus Typ 2 ist heute die am weitesten verbreitete Krankheit in der westlichen Welt, doch über die Ursachen weiss man noch sehr wenig. Genetische Defekte führen im Zusammenspiel mit Umwelteinflüssen und der Regulation durch posttranslationale Modifikationen auf Proteinebene über die Insulinresistenz zu Diabetes mellitus Typ 2. Die Rolle posttranslationaler Modifikationen bei der Entstehung der Insulinresistenz ist nur zu einem geringen Teil aufgeklärt. Doch zum Verständnis dieser Krankheit und als Ansatzpunkt für denkbare pharmakologische Therapien ist es notwendig, die molekularbiologischen Ursachen aufzuklären. Die Erforschung der Modulation der Insulinsignalweiterleitung durch Serin- und Threoninkinasen-vermittelte Phosphorylierungen an Insulinrezeptor-Substrat-1 (IRS-1) ist ein Schlüssel zur Aufklärung der zugrundeliegenden Pathomechanismen. Vor kurzem wurde gezeigt, dass IRS-1 ein Substrat der Proteinkinase (PKC)-zeta ist. Die resultierende Ser-/Thr-Phosphorylierung von IRS-1 reguliert die Insulinsignalweiterleitung unter hyperinsulinämischen Bedingungen. Zur Aufklärung der molekularen Mechanismen dieser feed-back-Regulation wurde im Rahmen dieser Arbeit ein in vitro-Phosphorylierungssystem entwickelt, um bakteriell exprimierte GST-Fusionsproteine von IRS-1 durch PKC-Isoformen zu phosphorylieren. Detektion der Phosphorylierung erfolgte durch eine neu entwickelte alkalische Negativionen Scan Technik. Die Sequenzierung der Phosphopeptide wurde mit Hilfe einer Ionenfalle durchgeführt. Im Fusionsprotein GST-IRS-1 N2 (Aminosäurensequenz 265-522 der cDNA von IRS-1) wurden Ser318 und Ser436 als in vitro-Phosphorylierungsstellen der PKC-Isoenzyme zeta...

IN VIVO PHOSPHORYLATION OF BACTERIAL–TYPE PHOSPHOENOLPYRUVATE CARBOXYLASE FROM DEVELOPING CASTOR OIL SEEDS AT THREONINE-4 AND SERINE-451

DALZIEL, Katie
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado
EN; EN
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36.41%
Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled anaplerotic enzyme situated at a pivotal branchpoint of plant C-metabolism. Plant genomes encode several closely related plant-type PEPC (PTPC) isozymes, and a distantly related bacterial-type PEPC (BTPC). Two physically and kinetically distinct oligomeric classes of PEPC occur in the endosperm of developing castor oil seeds (COS). Class-1 PEPC is a typical homotetramer composed of 107-kDa PTPC subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octameric complex arises from a tight interaction between Class-1 PEPC and 118-kDa bacterial-type PEPC (BTPC) subunits. BTPC functions as a catalytic and regulatory subunit of the allosterically-desensitized Class-2 PEPC, hypothesized to support PEP-flux to malate for leucoplast fatty acid synthesis. Previous studies established that BTPC: (i) subunits of COS Class-2 PEPC are phosphorylated at multiple sites in vivo and (ii) phosphorylation at Ser425 provides a new tier of enzyme control in developing COS. LC MS/MS and LTQ-FT MS identified Thr4 and Ser451 as additional in vivo phosphorylation sites of immunopurified COS BTPC (corresponding to acidophilic and basophilic protein kinase consensus sequences, respectively). Immunoblots probed with a phosphorylation-site specific antibody raised against a synthetic phosphopeptide indicated that Ser451 phosphorylation is promoted during seed development...

Régulation de la kinase Ste20 Slik de Drosophila par phosphorylation de la boucle d'activation

Panneton, Vincent
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
FR
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36.41%
Les kinases constituent une famille majeure de protéines qui régulent divers processus par la phosphorylation de leurs substrats, mais aussi par leur activité non- catalytique. Ce rôle indépendant de l’activité kinase a été observé chez quelques protéines dont des membres de la famille Sterile-20. La kinase Ste20 Slik de Drosophila aide au maintien de l’intégrité des tissus épithéliaux en phosphorylant l’ERM Moesin et peut aussi induire une prolifération cellulaire non-autonome indépendamment de son activité catalytique. La méthode de régulation de ces deux rôles était jusqu’ici inconnue. Nous avons identifié 19 sites de phosphorylation chez Slik par spectrométrie de masse. À l’aide de mutants, nous démontrons que les deux fonctions de Slik sont régulées par la phosphorylation d’au moins 2 résidus conservés de son segment d’activation par un mécanisme d’auto- et/ou trans-phosphorylation. Cette étude amène une meilleure compréhension de la régulation de l’intégrité épithéliale et de la croissance, deux processus clés qui sont souvent déréglés dans le cancer et certaines maladies génétiques.; Kinases constitute a major protein family which regulate diverse pathways through the phosphorylation of their substrates...

Importance de la phosphorylation de la ligase Itch dans la reconnaissance et l'ubiquitylation des protéines à domaine SH3

Forget, Rachel
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
FR
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36.48%
Itch est une ligase de l’ubiquitine impliquée dans la reconnaissance et la dégradation des protéines par le protéasome. Itch contient trois sites phosphorylés par JNK et il a été démontré que la phosphorylation de ces résidus est nécessaire pour que Itch puisse reconnaître et ubiquityler les protéines c-Jun et JunB. Ces sites de phosphorylation se retrouvent dans le domaine PRD responsable des interactions de Itch avec les protéines à domaine SH3. Si la phosphorylation de Itch par JNK est importante pour réguler son activité avec c-Jun et JunB, on connaît peu de choses sur les interactions de Itch avec les protéines à domaine SH3 ainsi que l’implication de la phosphorylation dans leur régulation. Nous avons donc créé des mutants de Itch par mutagenèse dirigée où les sites de phosphorylation étaient remplacés par des alanines (mutant non phosphorylable) et où l’un des trois sites était remplacé par un acide aspartique (mutant constitutivement phosphorylé). Ces mutants sont utilisés dans des tests d’interaction et d’ubiquitylation, dans le but de déterminer l’impact de la phosphorylation de Itch dans la reconnaissance et l’ubiquitylation des protéines SH3. Nos résultats montrent que, contrairement au modèle proposé...

Linker histone post-translational modifications and effects of phosphorylation on secondary structure and chromatin aggregation

Lopez Ramos, Rita
Fonte: [Barcelona] : Universitat Autònoma de Barcelona, Publicador: [Barcelona] : Universitat Autònoma de Barcelona,
Tipo: Tesis i dissertacions electròniques; info:eu-repo/semantics/doctoralThesis; info:eu-repo/semantics/publishedVersion Formato: application/pdf
Publicado em //2014 ENG
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Les histones linker juguen un paper important en l'organització i manteniment de la cromatina en estructures d'ordre superior i en la regulació transcripcional. La histona H1 en vertebrats té una estructura característica en tres dominis: un domini N-terminal curt i flexible; un domini globular central; i un domini C-terminal llarg. Els dominis N- i C-terminals (CTD) són molt bàsics i es troben desestructurats en solució aquosa. La distribució de càrrega és bastant uniforme al llarg de tot el CTD. La interacció amb el DNA indueix un plegament total i estable del CTD en condicions fisiològiques, fet que permet classificar aquest domini en el grup de les proteïnes intrínsecament desordenades, on el plegament i la unió al lligand estan acoblades. La fosforilació post-traduccional del CTD de la H1 té efectes en l'estructura secundària de la proteïna i en la condensació del DNA. L'estructura secundària de la H10 sencera es va analitzar per espectroscòpia d'infrarroig. La H10, igual que el CTD aïllat, també es plegà degut a la interacció amb el DNA i l'estructura secundària també va ser modulada per fosforilació. El canvi estructural induït per la fosforilació va consistir en un increment de la quantitat d'estructura β...

Phosphorylation Bar Codes Induce Distinct Conformations and Functionalities of beta-Arrestin

Nobles, Kelly Nicole
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2010
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36.47%

Seven transmembrane spanning receptors (7TMRs), or G-protein coupled receptors (GPCRs), represent the largest and most ubiquitous of the several families of plasma membrane receptors and regulate virtually all known physiological processes in humans. The classical paradigm of signal transduction in response to 7TMR stimulation involves an agonist-induced conformational change of the receptor which leads to interaction with and dissociation of the heterotrimeric G-protein into independent Galpha and Gbeta;gamma signaling subunits. Following their activation, 7TMRs are phosphorylated by G-protein coupled receptor kinases (GRKs) and subsequently recruit beta-arrestins. beta-arrestins are multifunctional adaptor proteins which not only desensitize G-protein signals, but also facilitate receptor internalization and mediate numerous signaling pathways on their own. As beta-arrestins universally interact with members of the 7TMR superfamily, we (1) developed an in vitro model system to assess conformational changes that occur in beta-arrestins in response to phosphorylation and (2) to map the sites of phosphorylation on the beta2 adrenergic receptor by different GRKs which would determine the conformation(s) assumed by beta-arrestin and thereby...

Integrative bioinformatics for kinase-concentric phosphorylation networks in Arabidopsis thaliana

Lv, Mengxi
Fonte: University of Delaware Publicador: University of Delaware
Tipo: Tese de Doutorado
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36.41%
Wu, Cathy; Lee, Jung-Youn; Phosphorylation, mediated by various types of protein kinases, is one of the most well studied mechanisms regulating signal transduction. The extensive studies produce a large amount of phosphorylation data, which are scattered and distributed in scientific articles and databases. However, lack of integrative PTM resources, especially in plants, makes it challenging for biological scientists to connect those fragmented data for systematic understanding and global analysis of PTMs. Here we developed an integrative bioinformatics approach combining text mining, data mining, protein ontology, visualization, and analysis methods, thereby facilitating PTM information integration and knowledge discovery in plants. For Arabidopsis thaliana , we identified a list of the top 15 most literature-documented kinase names through RLIMS-P, a text-mining tool to extract phosphorylation information. The resulting kinase list included PDK1 and PID, which are two kinases involved in a wide range of cellular activities and auxin polar transport respectively. Text-mining results demonstrated that phosphorylation of PID by PDK1 is a primary step to activate PID kinase's regulatory role in auxin polar transport. Moreover, given that 3 key Brassinosteroid (BR)-signaling components...

Structural effects of phosphorylation and beta-O-GlcNAcylation on alpha-helices and structural effects of phosphorylation and R406W on tau395-411

Elbaum, Michael Birch
Fonte: University of Delaware Publicador: University of Delaware
Tipo: Tese de Doutorado
Relevância na Pesquisa
36.48%
Zondlo, Neal; The dynamic interplay between phosphorylation and beta-O-GlcNAcylation (OGlcNAc) of serine and threonine plays critical roles in numerous intracellular processes. Changes in phosphorylation and OGlcNAcylation are linked to Alzheimer's disease, diabetes, and cancer. We have conducted a systematic study on a model alpha-helix to determine the structural effects of phosphorylation and OGlcNAcylation of serine and threonine residues, on the N-terminus, C-terminus, and internal positions (Ac-XKAAXAKAAXAKAAGY-NH 2 , Ac-YGAKAAAAKAAAAKAX-NH 2 ). We found that both phosphorylation and OGlcNAcylation on the N-terminus increase alpha-helix stability, with phosphorylation exhibiting a greater increase in alpha-helix stability than OGlcNAcylation. These stabilizing effects were found to be greater for threonine than serine, and for the dianionic phosphate over the monoanionic phosphate. In contrast, both phosphorylation and OGlcNAcylation reduced helix stability on internal and C-terminal positions relative to serine or threonine. These effects are not simply electrostatic interactions; we observe a unique cyclization of serine and threonine residues due to an intra-residue phosphate-amide hydrogen bond. Furthermore this interaction is greater for threonine than serine and for the dianionic phosphate over the monoanionic phosphate. On the N-terminus NMR data are consistent with an alpha-helix capping mechanism in which the phosphate hydrogen bonds to its own amide...

Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-1 in nerve terminals

Xue, Jing; Milburn, Peter J; Hanna, Bernadette; Graham, Mark E; Rostas, John A P; Robinson, Phillip J
Fonte: Portland Press Publicador: Portland Press
Tipo: Artigo de Revista Científica
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The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.