Signaling pathways are complex and function to transmit signals from the extracellular environment into the cell. Analysis of results obtained from a high throughput siRNA screen led to the identification of Membrane protein palmitoylated 3 (MPP3) and Leukocyte Tyrosine Kinase (LTK) as novel negative regulators of the Wnt pathway. MPP3 is a MAGUK family protein and domain mapping studies indicated that the Guk domain plays a role in the negative regulation of the pathway. LTK, a receptor tyrosine kinase, has several transcript variants one of which lacks the entire kinase domain (LTK∆KD). While LTK∆KD interacted with the Wnt receptor Frizzled7, the full length LTK did not, suggesting distinct modes of pathway regulation. Analysis of neuronal cells, NIE115 and Neuro2a, demonstrated LTK is expressed and that cells are Wnt3a responsive, thereby providing a neuronal model system appropriate for further studies on the mechanism and biological role of LTK as a negative regulator of the Wnt pathway

Signaling pathways are complex and function to transmit signals from the extracellular environment into the cell. Analysis of results obtained from a high throughput siRNA screen led to the identification of Membrane protein palmitoylated 3 (MPP3) and Leukocyte Tyrosine Kinase (LTK) as novel negative regulators of the Wnt pathway. MPP3 is a MAGUK family protein and domain mapping studies indicated that the Guk domain plays a role in the negative regulation of the pathway. LTK, a receptor tyrosine kinase, has several transcript variants one of which lacks the entire kinase domain (LTK∆KD). While LTK∆KD interacted with the Wnt receptor Frizzled7, the full length LTK did not, suggesting distinct modes of pathway regulation. Analysis of neuronal cells, NIE115 and Neuro2a, demonstrated LTK is expressed and that cells are Wnt3a responsive, thereby providing a neuronal model system appropriate for further studies on the mechanism and biological role of LTK as a negative regulator of the Wnt pathway

Activated receptor tyrosine kinases recruit many signalling proteins to initiate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K), a heterodimer consisting of a p85 regulatory protein and a p110 catalytic protein. Our laboratory has previously shown the p85α protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a p85α protein containing an arginine to alanine substitution at position 274 (p85R274A) that affects its GAP activity, caused sustained levels of activated platelet-derived growth factor receptors (PDGFRs), enhanced downstream signalling, and resulted in cellular transformation. Together with other data, this suggested that in p85R274A-expressing cells, PDGFRs are more rapidly trafficked through the endocytic pathway, which reduces opportunities for sorting events necessary for receptor degradation. Our laboratory has observed previously that p85 was capable of binding to both Rab5-GDP, as well as Rab5-GTP, which is an atypical characteristic of GAP proteins, whereas p110β had previously been reported to bind Rab5-GTP selectively. Based on these observations, this thesis project was designed to test the hypothesis that both proteins contributed GAP activity towards Rab5, with p85 providing a catalytic arginine residue (R274) and p110β providing switch stabilization functions specific to the GTP-bound state. To accomplish the thesis objective, cells expressing individual p85 defects (lacking GAP activity, R274A; or lacking p110-binding ability through deletion of residues 478-513, Δ110) were compared to cells expressing a double mutant missing both functions. Stable clonal NIH 3T3 cell lines were generated and selected in G418 and clones expressing similar levels of FLAG-tagged p85 wild type or mutants compared to the control cell lines (NIH 3T3, FLAG-vector control, p85 wild type, and p85R274A) were chosen for analysis. A time-course of PDGF stimulation showed that cells expressing p85R274A or p85Δ110+R274A have sustained phosphorylation levels of the PDGFR, reduced rates of PDGFR degradation and sustained MAPK/Erk signalling. Contrary to the cellular transformation previously reported for p85R274A-expressing cells, expression of p85Δ110+R274A did not lead to cellular transformation. These divergent results suggest that p85-associated p110 serves two functions. As the catalytic subunit of PI3K, one function is the localized generation of PI3,4,5P3 lipids at the plasma membrane for Akt activation, and possibly during receptor endocytosis where it could impact MAPK/Erk activation/deactivation kinetics and cell transformation. These results support a second function for p110 in the regulation of PDGFR activation/deactivation kinetics and PDGFR half-life, both strongly influenced by alterations in PDGFR trafficking. This suggests that p110β may regulate PDGFR trafficking by providing Rab5-GTP switch stabilization that complements the catalytic arginine residue (R274) within p85, and that p85α and p110β work together as a Rab5 GAP. The role of PDGFR in the localization of the RabGAP function of p85 to specific subcellular compartments was also examined. It was hypothesized that PDGFR may help localize the RabGAP function of p85 to vesicles containing Rab5 or Rab4 through the binding of p85 to phosphorylated tyrosine residues on activated PDGFR. Stable cell lines expressing individual p85 defects (lacking GAP activity, R274A; or lacking PDGFR-binding ability through site-directed mutation of residues 358 and 649 from arginine to alanine, ΔR; or a double mutant missing both functions) demonstrated that p85R274A or p85ΔR+R274A expression leads to sustained PDGFR activation and signalling, and to delayed PDGFR degradation in response to PDGF stimulation. The sustained signalling observed resulted in cellular transformation in cells expressing p85R274A or p85ΔR+R274A. The data suggests that PDGFR does not play a role in the localization of the RabGAP activity of p85. The findings of this study elucidates important non-canonical functions of the PI3K heterodimer and contributes to our understanding of how specific mutations in both p85 and p110β within regions implicated in the regulation of RabGAP activity can alter signalling events and lead to enhancement of tumour-associated phenotypes.

Aberrant subcellular localization of mutant transmembrane receptors is increasingly acknowledged as a possible mechanism for an altered signaling quality leading to transformation. There is evidence that mutated receptor tyrosine kinases of subclass III, for example the platelet-derived growth factor receptor (PDGFR) and KIT-protein, are aberrantly localized in human cancers. In order to further analyze this phenomenon, we investigated the localization of FLT3, a subclass III receptor tyrosine kinase frequently mutated in leukemia. By immunofluorescence staining and confocal laser scanning microscopy we found that in retrovirally transduced COS7 cells, wild type FLT3 receptor protein is localized primarily at the cell surface. In contrast, a mutant FLT3 receptor protein with an internal tandem duplication (ITD) accumulates in a perinuclear region and is not detectable at the plasma membrane. Surprisingly, and in contrast to previously published data, intracellular FLT3-ITD accumulation could neither be detected in the endoplasmic reticulum (ER) nor in the Golgi apparatus. Furthermore, transient overexpression per se leads to accumulation of wild type FLT3 receptor protein in the ER in addition to surface localization, probably due to inefficient intracellular transport by the overloaded sorting machinery of the secretory pathway. Based on our data and the immature glycosylation pattern of FLT3-ITD, we speculate that the mutant protein resides most probably in an unidentified compartment of the secretory pathway between the ER and the Golgi apparatus. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

L’HGF/SF (Hepatocyte Growth Factor/Scatter Factor) est le ligand du Récepteur Tyrosine Kinase (RTK) MET. Ce couple ligand-récepteur joue un rôle essentiel dans de nombreux processus biologiques tels que l’embryogenèse, la régénération tissulaire et l’angiogenèse. Comme pour de nombreux RTK, la dérégulation de l’activité de MET est associée à la progression et l’invasion tumorales. Bien que le récepteur MET ait été intensivement étudié au cours de ces dernières décennies, les processus moléculaires conduisant à son activation par l’HGF/SF restent encore mal connus et controversés.NK1, un variant naturel de l’HGF/SF, comprenant la partie N-terminale (N) et le premier domaine kringle (K1) de l’HGF/SF, possède une activité agoniste. En effet, NK1 dimérise spontanément en position « tête-bêche » et est considéré aujourd’hui comme la structure minimale permettant la dimérisation de MET et son activation. Afin de déterminer leur contribution respective, les domaines N et K1 isolés ont été produits par voie recombinante et ne montrent aucune ou qu’une très faible activité agoniste respectivement. Une présentation monovalente de ces domaines au récepteur MET ne semble donc pas pertinente pour déterminer leur fonction.Par conséquent, nous avons souhaité générer des complexes multivalents mimant le positionnement des domaines N et K1 au sein du dimère naturel. En tirant partie de la « One-Pot SEA ligation » développée au laboratoire, ces domaines ont été synthétisés par voie chimique et fonctionnalisés avec une extrémité C-terminale biotinylée (NB et K1B). En utilisant la streptavidine (S) comme plateforme de multimérisation, nous avons généré des complexes semi-synthétiques NB/S et K1B/S et déterminé les propriétés biologiques de ces nouvelles constructions multivalentes.L’ensemble des analyses de signalisations cellulaires et phénotypiques démontre sans équivoque que le complexe K1B/S est capable de mimer les réponses biologiques induites par l’HGF/SF et son variant NK1. De plus, le complexe K1B/S, injecté dans la circulation systémique, déclenche la signalisation de MET dans le foie. L’utilisation de ce complexe K1B/S nous a permis de démontrer que deux domaines K1, correctement assemblés et orientés, constituent l'interface minimale et suffisante requise pour déclencher une pleine activation de MET. A l’inverse, les premières données fonctionnelles ont démontré que le complexe NB/S ne lie pas directement MET mais utilise les héparanes sulfates comme pont moléculaire.Ces études utilisant de nouvelles configurations structurales pourraient donc servir de modèle de base au développement de nouveaux agonistes de MET dans le cadre de thérapies régénératives ou préservatrices, mais aussi d’antagonistes dans le cadre de thérapies anticancéreuses ciblées. / Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor tyrosine kinase (RTK) MET play an essential role in embryogenesis, tissue regeneration and angiogenesis. As observed for many others RTK, MET is also strongly involved in tumor progression and invasion mechanisms. Although numerous biological and structural approaches have been focused on the molecular processes leading to MET activation by HGF/SF, the HGF/SF-MET interaction framework remains only partially understood due to the complexity of the multivalent ligand-receptor binding events.NK1, a naturally occurring splice variant of HGF/SF, comprising the N-terminal part and the first kringle domain (K1) of HGF/SF, exhibits a partial agonistic activity toward MET. Indeed, in presence of heparan sulfates, NK1 self-associates into a “head-to-tail” dimer and is considered as the minimal structural module able to trigger MET dimerization and activation. Nevertheless, the individual role of N and K1 domains in the dimerization/activation of MET remain elusive.Stimulated by the conviction that monomeric N and K1 domains are not suitable for studying the functioning of HGF/SF-MET, we produced, by total chemical synthesis, biotinylated analogs of the N and K1 domains (NB and K1B). By combining with streptavidin (S), we engineered the semisynthetic constructs NB/S and K1B/S in order to determine the biological properties of these new multivalent architectures of N and K1 domains.In vitro, as observed with HGF/SF or NK1, we show that the K1B/S complex is able to fully activate MET signaling cascades to promote scattering, morphogenesis and survival phenotypes in various cell types. Even more, the K1B/S complex stimulates angiogenesis in vivo and, when injected systemically, triggers MET signaling in the liver. The use of this K1B/S complex allows us to demonstrate that two K1 domains, correctly assembled and oriented, constitute the minimal unit for sufficient MET activation. In contrast, first in vitro data have demonstrated that NB/S complex does not bind directly MET as previously thought, but rather, uses heparan sulfates as a molecular bridge.We envision these new structural configurations serving as a template for both the rational design of potent MET agonists (e.g. using K1B/S for regenerative therapies) and antagonists (e.g. using NB/S for targeted cancer therapies).

CSF-1R (colony-stimulating factor 1 receptor) est un récepteur transmembranaire à activité tyrosine kinase exprimé à la surface des monocytes, des macrophages et de leurs progéniteurs. Son ligand, CSF-1, oriente les cellules souches hématopoïétiques vers le lignage myéloïde et permet la différenciation des monocytes en macrophages. Une localisation nucléaire de CSF-1R a été décrite dans certaines lignées tumorales, dans des tumeurs mammaires primitives et dans les macrophages murins. Dans le noyau de ces cellules, CSF-1R régulerait la phosphorylation de protéines nucléaires et l'expression de gènes de la prolifération. Nous avons identifié une localisation nucléaire de CSF-1R dans les monocytes primaires humains par différentes approches et différents anticorps. La forme nucléaire de CSF-1R correspond à la protéine entière monomérique qui est transportée depuis la membrane plasmique vers le noyau, de manière rétrograde, après activation par son ligand et avec celui-ci. L'utilisation d'inhibiteurs de l'activité kinase de CSF-1R diminue la quantité de récepteur dans le noyau. En revanche le blocage des mécanismes d'export nucléaire dépendant de CRM1 par la leptomycine B conduit à l'accumulation de la protéine dans ce compartiment. Dans les monocytes, CSF-1R est localisé sur la chromatine, dans les régions intergéniques et introniques et colocalise avec la marque H3K4me1 présente au niveau des enhancers activés. CSF-1R est situé à proximité de gènes régulant la morphogénèse, le développement du système nerveux, l'ossification et la différenciation cellulaire. Le récepteur est présent sur le promoteur du gène PU.1, facteur de transcription clé dans la différenciation myéloïde et la génération des monocytes, ainsi que sur des gènes impliqués dans la différenciation, la polarisation, la survie et les fonctions des macrophages. Au niveau de la chromatine, CSF-1R interagit avec des facteurs de transcription comme EGR1 sur lequel il exerce un effet co-répresseur. Cette localisation nucléaire de CSF-1R est conservée lorsque les monocytes se différencient en macrophages en réponse à CSF-1. CSF-1R nucléaire est alors relocalisé vers les régions promotrices et exoniques où il colocalise avec la marque H3K4me3. Il est présent à proximité de gènes régulant la vascularisation, la phagocytose, le métabolisme, la réponse au stress et à l'hypoxie. Il interagit avec les facteurs de transcription ELK1 et YY1, et joue un rôle de co-activateur. Lorsque les monocytes sont différenciés en macrophages par une autre cytokine, le GM-CSF, CSF-1R reste dans le noyau des cellules mais sa localisation sur la chromatine et ses interacteurs diffèrent de ceux des monocytes et des macrophages générés par CSF-1, démontrant un régulation différentielle de CSF-1R nucléaire selon le stade de différenciation et les signaux environnementaux. Dans des monocytes de patients atteints de leucémie myélomonocytaire chronique, l’expression, la localisation sur l’ADN et les interacteurs de CSF-1R sont modifiés, indiquant une dérégulation des fonctions nucléaires du récepteur en condition pathologique. CSF-1R est donc localisé dans le noyau des monocytes et des macrophages où il exerce un rôle de régulation de l'expression des gènes dont PU.1. Des résultats préliminaires suggèrent une localisation nucléaire du récepteur dans certaines populations de progéniteurs myéloïdes où il pourrait participer à la regulation de la différenciation. De nombreux inhibiteurs de CSF-1R sont en développement afin de cibler les macrophages infiltrant les tumeurs. Nos résultats démontrent que certains inhibiteurs ont la capacité de cibler la forme membranaire et la forme nucléaire du récepteur et donc d'inhiber l'ensemble des activités de CSF-1R dans les cellules, renforçant l'activité potentielle de ces traitements. / CSF-1R (colony-stimulating factor 1 receptor) is a transmembrane receptor with a tyrosine kinase activity. It is expressed at the cell surface of monocytes, macrophages and their progenitors. Its ligand, CSF-1, has an instructive role on hematopoietic stem cells to direct their differentiation into the myeloid lineage. CSF-1R is also able to differentiate monocytes into macrophages. A nuclear location was described for CSF-1R in cancer cell lines, primary breast tumors and murine macrophages. In the cell nucleus, CSF-1R was suggested to regulate nuclear protein phosphorylation and gene expression. We demonstrate that a small part of CSF-1R is in the nucleus of primary human monocytes, using different antibodies and technical approaches. Nuclear CSF-1R corresponds to full length monomeric receptor. After activation by its ligand, CSF-1R is translocated form cell surface to the nucleus through a retrograde transport, together with CSF-1. Kinase activity inhibitors impaired this process while inhibitors of CRM1-dependant nuclear export (leptomycin B) can revert this effect. In monocytes, CSF-1R is localized on chromatin, mainly on intergenic and intronic regions. It colocalizes with H3K4me1 mark which signs active enhancers. The receptor is present around genes involved in morphogenesis, nervous system development, ossification and cell differentiation. CSF-1R is also located on PU.1 promoter, which is a master transcription factor involved in myeloid and monocyte differentiation. CSF- 1R is also present on genes implicated in macrophage functions, differentiation, polarization and survival. At the chromatin level, CSF-1R interacts with different transcription factors like EGR1 and exerts a co-repressive role to decrease or limit gene expression. CSF-1R nuclear localization persists in macrophages generated by exposure of monocytes to CSF-1. It entails CSF-1R relocalization on promoter-TSS and exonic regions where it colocalizes with H3K4me3 mark. The receptor is close to genes regulating vascularization, phagocytosis, metabolism, stress and hypoxia responses. CSF-1R interacts with ELK1 and YY1 to promote macrophage functions. When monocytes are differentiated into macrophages with GM-CSF, CSF-1R also remains in the nucleus. However, its chromatin localization and interactions change compared to monocytes and CSF-1 differentiated macrophages. This indicates that nuclear CSF-1R is differentially regulated, depending on the cytokine that triggers cell differentiation. In monocytes from chronic myelomonocytic leukemia, CSF-1R expression, chromatin localization and interactors are modified, indicating a deregulated CSF-1R nuclear function under pathological state. Altogether, we showed that CSF-1R is localized in the nucleus of human monocytes and macrophages where it regulates gene expression including PU.1. Preliminary results suggest CSF-1R nuclear location in myeloid progenitor subsets where the receptor could directly regulate the expression of myeloid differentiation genes. Targeting CSF-1R is currently tested as a therapeutic strategy to impair tumor infiltrating macrophages. Our results show that CSF-1R inhibitors are able to target both membrane and nuclear forms and thus to inhibit all CSF-1R activities in the cells, enhancing the potential therapeutic effects of these molecules.

Aberrant subcellular localization of mutant transmembrane receptors is increasingly acknowledged as a possible mechanism for an altered signaling quality leading to transformation. There is evidence that mutated receptor tyrosine kinases of subclass III, for example the platelet-derived growth factor receptor (PDGFR) and KIT-protein, are aberrantly localized in human cancers. In order to further analyze this phenomenon, we investigated the localization of FLT3, a subclass III receptor tyrosine kinase frequently mutated in leukemia. By immunofluorescence staining and confocal laser scanning microscopy we found that in retrovirally transduced COS7 cells, wild type FLT3 receptor protein is localized primarily at the cell surface. In contrast, a mutant FLT3 receptor protein with an internal tandem duplication (ITD) accumulates in a perinuclear region and is not detectable at the plasma membrane. Surprisingly, and in contrast to previously published data, intracellular FLT3-ITD accumulation could neither be detected in the endoplasmic reticulum (ER) nor in the Golgi apparatus. Furthermore, transient overexpression per se leads to accumulation of wild type FLT3 receptor protein in the ER in addition to surface localization, probably due to inefficient intracellular transport by the overloaded sorting machinery of the secretory pathway. Based on our data and the immature glycosylation pattern of FLT3-ITD, we speculate that the mutant protein resides most probably in an unidentified compartment of the secretory pathway between the ER and the Golgi apparatus. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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Bourse de maîtrise du Groupe de recherche sur le système nerveux central GRSNC, (2009,2010) Bourse d’études supérieures du Canada Frederick Banting et Charles Best, IRSC Instituts de recherche en santé du Canada, (2011) / EphA7 est un membre de la famille des récepteurs à tyrosine kinase Eph, qui régulent l’adhérence et la motilité cellulaires. EphA7 est hautement conservé chez les vertébrés et largement exprimé durant l'embryogenèse, en particulier pendant le développement du SNC. Dans le cerveau adulte, EphA7 est transcrit principalement dans l'hippocampe, avec de faibles niveaux d'expression ailleurs. Nous avons cartographié sa distribution dans le cerveau du rat et de la souris adultes, par hybridation in situ et immunohistochimie en microscopie photonique et électronique. Les deux méthodes montrent une distribution de marquage très cohérente. Le signal le plus fort a été observé dans l’hippocampe, avec des niveaux moins élevés dans l’habénula, le striatum, l’amygdale, les cortex cingulaire, piriforme et entorhinal, ainsi que le cervelet. Au niveau ultrastructural, dans l’hippocampe, l’immunoréactivité d’EphA7 a été localisée dans le cytoplasme des cellules granulaires (gyrus dentelé) et pyramidales (CA1 et CA3) en ordre décroissant d’intensité. Dans le neuropile de CA1, des épines dendritiques et des prolongements astrocytaires, souvent périsynaptiques, ont été les éléments le plus fréquemment marqués. Plus rarement, nous avons aussi rencontré des dendrites et des terminaisons axonales immunopositives. La localisation préférentielle d’EphA7 dans les épines dendritiques et les prolongements astrocytaires périsynaptiques est conséquente avec un rôle de ce récepteur dans la plasticité synaptique / Abstract: EphA7 is a member of the family of Eph receptor tyrosine kinases, which regulate cell adhesion and motility. EphA7 is highly conserved in vertebrates and widely expressed during embryogenesis, especially during the development of the CNS. In the adult brain, EphA7 is transcribed mainly in the hippocampus, with low expression levels elsewhere. We have mapped its distribution in the adult brain of rat and mice by in situ hybridization and by immunohistochemistry in light and electron microscopy. Both methods show very consistent labelling distribution. The strongest signal was observed in the hippocampus, but modest levels were detected in the habenula, striatum, amygdala, the cingulate, piriform and entorhinal cortex, and the cerebellum. At the ultrastructural level, in the hippocampus, EphA7 immunoreactivity was localized in the cytoplasm of granule (dentate gyrus) and pyramidal cells (CA1 and CA3) in descending order of intensity. In the neuropil of CA1, dendritic spines and astrocytic processes, often perisynaptic were the most frequently labelled. More rarely, we also observed immunopositive dendrites and axon terminals. The preferential localization of EphA7 in dendritic spines and perisynaptic astrocytic processes is consistent with a role of this receptor in synaptic plasticity