2024-03-28T10:16:17Zhttps://www.tdx.cat/oai/requestoai:www.tdx.cat:10803/34802017-08-29T21:54:32Zcom_10803_120col_10803_127
TDX (Tesis Doctorals en Xarxa)
author
Riera Bonet, Marta
authoremail
mrbgmm@cid.csic.es
authoremailshow
true
director
Pagès i Torrens, Montserrat
2011-04-12T14:18:04Z
2003-02-12
2002-09-16
8468802115
http://www.tdx.cat/TDX-0212103-193735http://hdl.handle.net/10803/3480
B.46.443-2002
La proteïna quinasa CK2 és una Ser/Thr quinasa ubiqua i altament conservada entre les espècies. S'ha trobat a pràcticament tots els compartiments de totes les cèl.lules eucariotes examinades. Tant en animals com en plantes s'ha demostrat que pot fosforilar in vitro un alt nombre de proteïnes involucrades en molts processos cel.lulars, no obstant, es desconeix el mecanisme de regulació de l'enzim així com la seva funció biològica. L'holoenzim CK2 és un heterotetràmer, format per dos tipus de subunitats, les catalítiques CK2a i les reguladores CK2b, encara que en blat de moro fins el moment de començar aquesta tesi no s'havia descrit l'existència de subunitats reguladores CK2b. Entre els substrats de CK2 descrits en blat de moro es troba la proteïna Rab17. Rab17 és una proteïna LEA (Late Embryogenesis Abundant) que s'acumula en embrió madur i en resposta a estrés abiòtic i àcid abscisic. Estudis funcionals previs varen indicar una possible funció de Rab17 en el procés de germinació en condicions d'estrés osmotic que podria està regulada per fosforilació per CK2.<br/><br/>Resultats<br/><br/>La tesi es divideix en tres capítols: En el primer es realitza la caracterització molecular de la proteïna quinasa CK2 de blat de moro i consta de dues publicacions internacionals: Maize protein kinase CK2: regulation and functionality of three beta subunits. Marta Riera, Giovanna Peracchia, Eulalia de Nadal, Joaquin Ariño and Montserrat Pagès. Plant J. 2001, 25: 365-374 i Distinctive features of plant protein kinase CK2. Marta Riera, Giovanna Peracchia, Montserrat Pagès. Mol. Cel. Biochem. 2001, 227:119-127. En aquests treballs es descriu l'aillament de tres subunitats reguladores CK2b en blat de moro i una nova subunitat catalítica. També s'estudia l'expressió gènica de les diferents subunitats CK2a/b, la especificitat d'interacció entre elles i es demostra la funcionalitat de les subunitats CK2b de blat de moro mitjançant estudis de complementació en llevat. També es va realitzar la purificació de l'holoenzim CK2 recombinant de blat de moro i es caracteritzar a nivell bioquímic, demostrant que presenta una estructura heteroteramèrica. En el segon capítol es presenta l'estudi de la regulació de la proteïna Rab17 per la proteïna quinasa CK2 de blat de moro. Es va determinar que existia interacció física entre les subunitats reguladores CK2b1 i CK2b3 i la proteïna Rab17 mitjançant l'aproximació del sistema del doble híbrid i l'anàlisi de "pull-down". També es va realitzar un estudi de la fosforilació in vitro i en extractes de blat de moro de la proteïna Rab17 pels diferents enzims CK2. A més, es va estudiar la localització subcel.lular tant de la proteïna Rab17 com de les subunitats CK2a/b fusionades a la proteïna GFP mitjançant transformació transient de cèl.lules de ceba i es va comprovar que existia una relocalització subcel.lular de Rab17 depenent de el seu estat de fosforilació CK2. També es va demostrar una localització nucleolar de les subunitats catalítiques CK2a mentre que les subunitats reguladores CK2b queden excloses d'aquest compartiment. Per últim al tercer capítol es descriu una sèrie de proteïnes que es van aïllar mitjançant un "screening" de doble híbrid utilitzant la subunitat reguladora CK2b1 com a "bait". Es tracta de proteïnes de localització nuclear implicades en diversos processos de regulació gènica com factors de transcripció de diferent tipus, proteïnes de reparació de DNA o d'unió a RNA indicant que la proteïna quinasa CK2 de blat de moro està implicada en processos essencials per a la viabilitat cel.lular.
Protein kinase CK2 is a ubiquitous and highly conserved Ser/Thr kinase present in nucleus and cytoplasm of all eukaryotic cells examined to date. It is not responsive to known second-messengers molecules and both in animals and plants is able to phosphorylate high number of proteins, however, its precise biological functions remain unknown. The enzyme is a heterotetramer composed of two types of subunits, catalytic (CK2a) and regulatory (CK2b), however, in maize, the existence of CK2 regulatory subunits was not previously described. It is known that maize Rab17 protein is in vitro phosphorylated by protein kinase CK2. Rab17 protein (for Responsive to ABA) is a Late Embryogenesis Abundant protein that accumulates during seed maturation and can be induced to reappear by ABA treatment or osmotic stress in the vegetative tissues. Preliminary functional studies indicate that a possible role of Rab17 in germination process under stress conditions should be regulated by CK2 phosphorylation.<br/><br/>Results<br/><br/>The thesis manuscript includes three chapters: The first chapter is about the molecular characterization of maize protein kinase CK2 and includes two international publications: Maize protein kinase CK2: regulation and functionality of three beta subunits. Marta Riera, Giovanna Peracchia, Eulalia de Nadal, Joaquin Ariño and Montserrat Pagès. Plant J. 2001, 25: 365-374 and Distinctive features of plant protein kinase CK2. Marta Riera, Giovanna Peracchia, Montserrat Pagès. Mol. Cel. Biochem. 2001, 227:119-127. These two works demonstrates the existence and functionality of three CK2b regulatory subunits and a novel catalytic CK2a subunit in maize. It has been also studied the expression pattern of all CK2a/b subunits and the specific interactions between the different subunits. Moreover, it was demonstrated the functionality of maize CK2b regulatory subunits by yeast complementation assays. Purification and biochemical characterization of recombinant maize CK2 demonstrate the heterotetrameric structure of maize holoenzyme. In the second chapter is studied the regulation of Rab17 by CK2 phosphorylation. By using two-hybrid system and pull-down assays is showed that Rab17 interacts with regulatory subunits CK2b1 and CK2b3. It was also studied the phosphorylation of Rab17 by different CK2 enzymes. It was analysed in detail by confocal microscopy the subcellular localization of Rab17 and CK2a/b subunits fused to green-fluorescence protein (GFP) in transformed onion cells, showing that the subcelular localization of Rab17 is dependent on its CK2 phosphorylation . It is also described a mainly nucleolar localization of CK2a catalytic subunits whereas the regulatory CK2b subunits are absents of this compartment. Finally, the third chapter contains the description of several proteins isolated by two-hybrid screening using the regulatory CK2b1 subunit as a bait. All proteins present nuclear localization and are involved in several processes of gene regulation such as transcription factors, DNA repair or RNA binding indicating that maize CK2 is involves in main mechanism essentials for cell survival.
spa
Rab17
CK2
Plantes
Caracterització molecular i funcional de la proteïna quinasa CK2 de blat de moro (Zea mays)
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