Etudes fonctionnelles de Tyk2 dans la voie de signalisation de l’IFNalpha : analyse d’un nouvel interacteur et d’une mutation activatrice

par Milica Gakovic

Thèse de doctorat en Physiologie et Physiopahologie

Sous la direction de Sandra Pellegrini.

Soutenue en 2007

à Paris 6 .


  • Résumé

    Les tyrosines kinases de la famille Jak (Tyk2, Jak1-3) sont impliquées dans la signalisation par les cytokines hélicoïdales. Une partie de ma thèse a porté sur l’étude de Pot1, un nouvel interacteur de Tyk2. Afin d’évaluer le rôle de Pot1 dans la voie de signalisation de l’IFN, j’ai mesuré la réponse à l’IFN de cellules déplétées en Pot1 en évaluant la phosphorylation des STATs et l’induction d’un gène rapporteur. La diminution de l'expression de Pot1 n’a pas d’effet sur la signalisation par l’IFN. La deuxième partie de mon travail a porté sur l’étude de la substitution V678F dans la Tyk2. Nous avons établi, à partir de cellules Tyk2-déficientes, des clones stables exprimant la protéine sauvage ou mutante. Nous avons aussi analysé le mutant Tyk2V678F associé à un récepteur artificiel de type homodimérique. Nos résultats montrent que la mutation V678F augmente in vivo et in vitro la capacité de Tyk2 à s’auto-phosphoryler, cela plus fortement dans le cas du récepteur homodimérique. De plus, le mutant acquiert la capacité de phosphoryler STAT3 en absence de ligand mais n’affecte pas la réponse à l’IFN en terme de phosphorylation de Jak1 et STAT1/2.

  • Titre traduit

    Functional studies of Tyk2 in IFNalpha signaling : a new interactor and an activating mutation


  • Résumé

    Tyk2 is a member of the Jak family of tyrosine kinases (Jak1, Jak2, Jak3 and Tyk2), which are indispensable components of -helical cytokine signaling cascades. Receptors for - helical cytokines are mostly made of two transmembrane subunits that associate with Jaks. Ligand bridging of two receptor chains brings together the associated Jaks, enabling their activation by transphosphorylation. Activated Jaks phosphorylate the STATs (Signal Transducer and Activator of Transcription) which translocate into the nucleus to drive gene expression. The Jaks have an N-terminal FERM (band 4. 1-ezrin-radixin-moesin) domain, followed by an "SH2-like" domain and two kinase domains: a kinase-like (KL) domain and the catalytic tyrosine-kinase domain. The FERM and SH2-like domains are necessary for receptor binding. The KL domain has no catalytic activity, but plays an important regulatory role. The laboratory is particularly interested in the type I interferon (IFN/) receptor, made of two subunits IFNAR1 and IFNAR2, which bind Tyk2 and Jak1, respectively. During the first part of my thesis, I analyzed a new Tyk2 interacting protein, Pot1 (Partner of Tyk2). Pot1 was isolated in a yeast two-hybrid screen using the Tyk2 FERM domain as bait. To assess the role of Pot1 in IFNsignaling, I monitored IFN-induced response in Pot1-depleted cells by measuring STAT phosphorylation and the induction of a reporter gene. These experiments showed that, in this system, Pot1 depletion had no effect on IFN-induced signaling. A two-hybrid screen was performed with Pot1 as bait. Among the 14 proteins found with high interaction confidence, we focused on GIT1 (G protein-coupled receptor kinase interactor 1), an adaptor protein implicated in a number of cellular processes, like cell migration, receptor internalization and EGF and angiotensin II signaling. To analyze the role of GIT1 in IFNsignaling, I monitored IFN-induced receptor internalization, STAT phosphorylation and the induction of a reporter gene in GIT1-depleted cells. The results obtained allow us to exclude a role for GIT1 in type I IFN signaling. During the second part of my thesis, I analyzed the effect of the V678F substitution on Tyk2 function. This mutation, located in the KL domain, corresponds to the V617F mutation of Jak2 found at the origin of Polycythemia vera. To study the effect of the V678F mutation on Tyk2 activity, I reconstituted Tyk2-deficient cells with Tyk2 WT or the V678F mutant and monitored IFN-induced response. Our results show that the V678F mutation augments basal Tyk2 kinase activity measured in vitro. This gain-of-function leads to an increase of the basal STAT3 phosphorylation level, but has no effect on IFN-induced Jak1 and STAT1/2/5 phosphorylation. As opposed to Jak2, Tyk2 has been implicated only in signaling via heterodimeric receptor complexes. Interestingly, it has been shown that Jak2V617F needs the coexpression of a cognate homodimeric receptor to fully exert its transforming activity in the BaF3 cellular model system. Therefore, we analyzed the effect of Tyk2V678F on signaling via an artificial homodimeric receptor. To this end, we used Tyk2-deficient cells that express a chimeric receptor containing the extracellular domain of erythropoietin receptor fused to the intracellular region of IFNAR1. These cells were stably reconstituted with Tyk2WT or the V678F mutant. In this context, Tyk2V678F confers ligand hypersensitivity as seen by STAT1/3/5 phosphorylation. Moreover, the ensemble of these data point to STAT3 as a preferred substrate of Tyk2.

Consulter en bibliothèque

La version de soutenance existe sous forme papier

Informations

  • Détails : 1 vol. (129 f.)
  • Annexes : Bibliogr. f.108-120. 206 réf. bibliogr. Annexes

Où se trouve cette thèse ?

  • Bibliothèque : Université Pierre et Marie Curie. Bibliothèque Universitaire Pierre et Marie Curie. Section Biologie-Chimie-Physique Recherche.
  • Consultable sur place dans l'établissement demandeur
  • Cote : T Paris 6 2007 434
Voir dans le Sudoc, catalogue collectif des bibliothèques de l'enseignement supérieur et de la recherche.