Structural and functional interactions between cytokine receptors and JAK kinases / Interactions structurelles et fonctionnelles entre les récepteurs des cytokines et les JAK kinases

Le rôle des Janus kinases (JAKs) dans la signalisation induite par les cytokines a été longtemps considéré comme limité à l'initialisation de la signalisation, à la phosphorylation des récepteurs et de leurs partenaires. L'objectif principal de cette thèse était d'explorer les effets des JAKs sur le trafic et la stabilité du récepteur homodimérique de la thrombopoïétine (TpoR) et des récepteurs hétérodimériques pour l'IL-9 et l'IL-2. Nous avons démontré que le TpoR était plus exprimé, mais aussi plus stable à la surface cellulaire en présence d'une quantité supplémentaire de JAK2 ou de Tyk2. La séquence du domaine cytoplasmique du TpoR couvrant les régions Box1 et Box2 ainsi qu'un domaine N-terminal FERM intact sur les JAKs sont nécessaires à l'augmentation de la forme mature, résistante à l'endoglycosidase-H, du TpoR. L'augmentation du nombre de TpoR en surface est identique dans les cellules surexprimant JAK2 ou Tyk2, mais JAK2 est beaucoup plus puissante comme activateur de la signalisation. Néanmoins, en présence de Tpo, Tyk2 seul est aussi capable d'activer les différentes voies de signalisation à partir du TpoR. Le rôle des JAKs a également été évalué sur les complexes de récepteurs hétérodimériques pour l'IL-9 et l'IL-2. JAK1 augmente spécifiquement la localisation à la surface cellulaire de l'IL-9Ra et de l'IL-2Rb, alors que JAK3 augmente spécifiquement la localisation à la surface cellulaire de la sous-unité gc. A nouveau, la région cytoplasmique couvrant Box1-Box2 sur les récepteurs et le domaine FERM intact dans la portion N-terminale de JAK1 et de JAK3 sont nécessaires pour ces effets. Les JAKs augmentent la localisation à la surface cellulaire de ces récepteurs essentiellement en stimulant la maturation du récepteur, sans changer le niveau total d'expression de la protéine. De plus, en présence d'un niveau identique d'IL-9Ra en surface, une expression plus importante de JAK1 conduit à une augmentation de la sensibilité à l'IL-9. Des résultats similaires ont été obtenus pour le TpoR. En conclusion, les JAKs possèdent deux rôles différents sur les récepteurs des cytokines : elles induisent et amplifient la transduction du signal, mais elles modulent aussi le nombre de récepteurs des cytokines disponibles à la surface cellulaire. Cette modulation ajoute un niveau supplémentaire de régulation à la signalisation à partir des récepteurs des cytokines. Après stimulation à la Tpo, différents profils de phosphorylation des tyrosines sont induits dans les cellules surexprimant le TpoR et JAK2 ou Tyk2. Sur base de ces résultats, une collaboration avec la société Cell Signaling Technology a débuté. Cette compagnie a développé une méthode d'isolement des peptides phosphorylés sur tyrosines, suivie par le séquençage de ces phosphopeptides par spectrométrie de masse. Le but de cette collaboration était la découverte de nouveaux partenaires de signalisation de JAK2 ou de Tyk2, ainsi que de nouveaux sites de phosphorylation essentiellement dans les protéines JAK2 et Tyk2. Après stimulation à la Tpo, des sites de phosphorylation connus ont été détectés dans JAK2 et, dans les cellules surexprimant Tyk2, la tyrosine en position 292 (Tyr292) dans la séquence de Tyk2 s'est révélée être phosphorylée. Les résultats obtenus par mutation de ce résidu en phénylalanine indiquent que le groupement phosphate sur la Tyr292 régule négativement l'activité kinase de Tyk2.The role of the Janus kinases (JAKs) in cytokine signaling was for a long time thought to be limited to initiation of signaling, phosphorylation of the receptors and of their signaling partners. The aim of the thesis was to explore JAK effects on traffic and stability of the homodimeric receptor for thrombopoietin (TpoR) and of the heterodimeric receptor complexes for IL-9 and IL-2. We report the effect of two different JAKs, JAK2 and Tyk2, on stimulating TpoR cell surface localization and protein stability. Sequences encompassing Box1 and Box2 regions of TpoR cytosolic domain and an intact JAK N-terminal FERM domain are required for the increase of the mature, endoglycosidase-H resistant form of TpoR. Enhancement of TpoR cell surface localization is identical in JAK2 or Tyk2 overexpressing cells, but JAK2 is much more potent as signal activator. Nevertheless, Tyk2 alone is able to induce signaling via TpoR upon Tpo addition. The role of cognate JAKs was also assessed in the case of heterodimeric IL-9 and IL-2 receptor complexes. JAK1 specifically increases cell surface localization of IL-9Ra and of IL-2Rb, whereas JAK3 specifically enhances gc cell surface localization. Again, Box1-Box2 regions on the receptors and an intact N-terminal FERM domain in JAK1 and JAK3 are required for these effects. JAKs promote these receptor cell surface localizations mainly by increasing receptor maturation, without a change in total receptor protein levels. At equal cell surface levels of IL-9Ra, higher JAK1 expression level leads to enhanced IL-9-dependent signaling. Similar results were obtained for TpoR. In conclusion, JAKs have clearly two different roles on cytokine receptors : they induce and amplify signal transduction but they also modulate the number of cytokine receptors available at the cell surface. This modulation adds a new layer of regulation for cytokine receptor signaling. After Tpo stimulation, different tyrosine phosphorylation patterns in TpoR-Ba/F3 cells overexpressing JAK2 or Tyk2 are induced. Based on these results, a collaboration was initiated with the company Cell Signaling Technology. They have developed an immunoaffinity method of phosphotyrosine peptide isolation followed by mass spectrometry sequencing. We wanted to discover new signaling phosphorylated partners involved in interactions with JAK2 or Tyk2, as well as new phosphorylation sites especially in JAK2 and Tyk2 proteins. Upon Tpo stimulation, known phosphorylated sites were found in JAK2 and, in Tyk2 overexpressing cells, Tyk2 was discovered to be phosphorylated on tyrosine in position 292 (Tyr292). Results obtained by mutation of this residue into phenylalanine indicate that the phosphate group on Tyr292 negatively regulates Tyk2 kinase activity.Thèse de doctorat en sciences biomédicales (biologie cellulaire et immunologie)(SBIM 3) -- UCL, 200

High-throughput gateway bicistronic retroviral vectors for stable expression in mammalian cells: Exploring the biologic effects of STAT5 overexpression

Stable expression of cloned genes in mammalian cells has been achieved in the past by retroviral transduction using bicistronic retroviral vectors. In these vectors, the use of an Internal Ribosome Entry Site (IRES) allows simultaneous expression of a protein of interest and a fluorescence marker. However, traditional cDNA cloning in these vectors is often difficult. Here we report the construction of a high-throughput retroviral vector using the Invitrogen "Gateway" Cloning system. The Gateway recombination sequences (attR) flanking the ccdB and chloramphenicol resistance genes were incorporated at the 59 of the IRES of pMX-IRES-GFP, -CD2, or -CD4 vectors. Through recombination, these vectors can acquire cDNAs coding for genes of interest, which will result in simultaneous expression of the recombined gene and the marker protein. We constructed Gateway bicistronic vectors coding for the erythropoietin receptor (EpoR) and GFP, CD4, or CD2. Epo-dependent proliferation assays and analysis of Jak2-dependent EpoR cell-surface expression showed that these vectors were able to function indistinguishable from the original pMX-EpoR-IRES-GFP. The expression levels of the genes cloned upstream the IRES were proportional to the levels of expression of GFP, which was cloned downstream of the IRES. We used the same approach and generated Ba/F3 cells that overexpress STAT5a, STAT5b, or a constitutively active form of STAT5. Overexpression of STAT5 lead to a significant effect on the intrinsic adherence to plastic of these cells, but did not change their proliferative responses to cytokines. We discuss possible applications of the new vectors for cell signaling and expression cloning

JAK kinases overexpression promotes in vitro cell transformation

Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis. Here, we took advantage of the Ba/F3 cell line, a murine proB cell line dependent on IL-3 for growth, to analyse mechanisms of constitutive STAT activation in vitro. Cytokine-independent and tumorigenic Ba/F3 cell lines were derived from a two-step selection process. Cells transfected with a defective IL-9 receptor acquire IL-9 responsiveness during a first step of selection, and progress after a second selection step to autonomously growing tumorigenic cells. Microarray analysis pointed to JAK1 overexpression as a key genetic event in this transformation. Overexpression of JAK1 not only increased the sensitivity to IL-9 but also allowed a second selection step toward cytokine-independent growth with constitutive STAT activation. This progression was dependent on a functional FERM and kinase JAK1 domain. Similar results were observed after JAK2, JAK3 and TYK2 overexpression. All autonomous cell lines showed an activation of STAT5, ERK1-2 and AKT but only TYK2-overexpressing cell lines showed a constitutive activation of STAT3. Thus, JAK overexpression can be considered as one of the oncogenic events leading to the constitutive activation of the JAK-STAT pathway

Janus kinases affect thrombopoietin receptor cell surface localization and stability.

The thrombopoietin receptor (TpoR) regulates hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. TpoR signals by activating Janus kinases JAK2 and Tyk2. Here we show that, in addition to signaling downstream from the activated TpoR, JAK2 and Tyk2 strongly promote cell surface localization and enhance total protein levels of the TpoR. This effect is caused by stabilization of the mature endoglycosidase H-resistant form of the receptor. Confocal microscopy indicates that TpoR colocalizes partially with recycling transferrin in Ba/F3 cells. The interaction with JAK2 or Tyk2 appears to protect the receptor from proteasome degradation. Sequences encompassing Box1 and Box2 regions of the receptor cytosolic domain and an intact JAK2 or Tyk2 FERM domain are required for these effects. We discuss the relevance of our results to the reported defects of TpoR processing in myeloproliferative diseases and to the mechanisms of Tpo signaling and clearance via the TpoR

Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor α Homodimers*S⃞

Activating mutations in JAK1 have been reported in acute lymphoblastic leukemias, but little is known about the mechanisms involved in their constitutive activation. Here, we studied the ability of JAK1 V658F and A634D to activate the Janus kinase (JAK)/STAT pathway upon ectopic expression in HEK293 cells alone or together with the other components of the interleukin-9 receptor complex (IL-9Rα, γc, and JAK3). Expression of JAK1 mutants alone failed to trigger STAT activation, but co-expression of the IL-9Rα chain promoted JAK1 mutant phosphorylation and STAT activation. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Rα involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with a functional IL-9Rα to activate STAT factors. Several lines of evidence indicated that IL-9Rα homodimerization was involved in this process. IL-9Rα variants with mutations of the JAK-interacting BOX1 region not only failed to promote JAK1 activation but also acted as dominant negative forms reverting the effect of wild-type IL-9Rα. Coimmunoprecipitation experiments also showed the formation of IL-9Rα homodimers. Interestingly, STAT activation was partially inhibited by expression of γc, suggesting that overlapping residues are involved in IL-9Rα homodimerization and IL-9Rα/γc heterodimerization. Co-expression of wild-type JAK3 partially reverted the inhibition by γc, indicating that JAK3 cooperates with JAK1 mutants within the IL-9 receptor complex. Similar results were observed with IL-2Rβ. Taken together, our results show that IL-9Rα and IL-2Rβ homodimers efficiently mediate constitutive activation of ALL-associated JAK1 mutants

Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer

Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation. By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations. We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor. The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures. We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies

JAK2, the JAK2 V617F mutant and cytokine receptors

Recently, a unique recurrent somatic mutation was identified as a major molecular event in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Expression of this mutant in cytokine-dependent hematopoietic cell lines induces autonomous growth. This effect is enhanced by overexpression of cytokine receptors, and can be inhibited by co-expression at higher levels of the wild type JAK2, which may compete for a limited pool of receptors. In JAK2-deficient cells, we showed that JAK2 V617F can transmit signals ftom ligand-activated TpoR or EpoR. Furthermore, the mutant JAK2 can be demonstrated to stimulate traffic of the EpoR. Thus, JAK2 V617F mutant must be able to interact via its intact FERM-SH2 domains with the cytosolic domains of cytokine receptors. A synergy between JAK2 V617F and insulin-like growth factor 1 receptor (IGFIR) can be detected in cytokine-dependent cell proliferation. Once cells are rendered autonomous by expression of JAK2 V617F, IGF1 acquires the ability to activate the JAK-STAT pathway. Thus, expression of JAK2 V617F may explain the described hypersensitivity of PV erythroid progenitors to IGFI. The V617 is conserved in two other mammalian JAKs, JAK1 and Tyk2. The homologous mutants JAK1 V658F and Tyk2 V678F are also active in proliferation and transcriptional assays. Such mutants may be found in human cancers or autoimmune diseases. In contrast, the JAK3 M592F does not lead to activation of JAK3. Current hypotheses on how JAK2 V617F contributes to three myeloproliferative diseases, and which other events may favor one disease versus another, are discussed. (c) 2006 Elsevier Masson SAS. All rights reserved

Thrombopoietin receptor down-modulation by JAK2 V617F: restoration of receptor levels by inhibitors of pathologic JAK2 signaling and of proteasomes.

The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs