Feedback regulation of Gab2-dependent signaling by the Ras/MAPK pathway

La voie de signalisation des R��cepteurs Tyrosine Kinase (RTK) occupe un r��le central dans la r��gulation de la croissance cellulaire, la prolif��ration, la diff��rentiation et la motilit��. Une r��gulation anormale des RTKs m��ne �� plusieurs maladies humaines telles que le cancer du sein, la seconde cause de mortalit�� chez les femmes �� cause de l���amplification et la mutation fr��quente de la prot��ine tyrosine kinase HER2 (ERBB2). Grb2-associated binder (Gab) 2 est une prot��ine adaptatrice qui agit en aval de plusieurs RTKs, y compris HER2, pour r��guler de multiples voies de signalisation. En r��ponse �� la stimulation par de nombreux facteurs de croissances et cytokines, Gab2 est recrut�� �� la membrane plasmique o�� il potentialise l���activation des voies de signalisation Ras/mitogen-activated protein kinase (MAPK) et PI3K (phosphatidylinositol-3-kinase)/Akt (protein kinase B). En plus d���occuper un r��le essentiel durant le d��veloppement du syst��me h��matopo����tique, Gab2 est souvent amplifi�� dans les cancers, notamment le cancer du sein et les m��lanomes. Cependant, les m��canismes mol��culaires qui r��gulent le fonctionnement de Gab2 sont peu connus. Il est ��tabli que lors de l���activation des RTKs, Gab2 est phosphoryl�� au niveau de plusieurs r��sidus Tyrosine, menant �� l���association de diff��rentes prot��ines comme p85 et Shp2. En plus de la phosphorylation en Tyrosine, notre laboratoire ainsi que d���autres groupes de recherche avons identifi�� que Gab2 est aussi phosphoryl�� au niveau de r��sidus Ser/Thr suite �� l���activation de la voie de signalisation MAPK. Cependant, la r��gulation des fonctions de Gab2 par ces modifications post-traductionnelles est encore peu connue. Dans le but de comprendre comment Gab2 est r��gul�� par la voie de signalisation MAPK, nous avons utilis�� diff��rentes approches. Dans la premi��re partie de ma th��se, nous avons d��termin�� un nouveau m��canisme d��montrant que la voie de signalisation Ras/MAPK, par le biais des prot��ines kinases RSK (p90 ribosomal S6 kinase), phosphoryle Gab2. Ce ph��nom��ne se produit �� la fois in vivo et in vitro au niveau de trois r��sidus Ser/Thr conserv��s. Des mutations au niveau de ces sites de phosphorylation entrainent le recrutement de Shp2 menant �� l���augmentation de la motilit�� cellulaire, ce qui sugg��re que les prot��ines RSK restreignent les fonctions d��pendantes de Gab2. Ce ph��nom��ne est le r��sultat de la participation de RSK dans la boucle de r��troaction n��gative de la voie de signalisation MAPK. Dans la seconde partie de ma th��se, nous avons d��montr�� que les prot��ines ERK1/2 phosphorylent Gab2 au niveau de plusieurs r��sidus pS/T-P �� la fois in vitro et in vivo, entrainant l���inhibition du recrutement de p85. De plus, nous avons ��tabli pour la premi��re fois que Gab2 interagit physiquement avec ERK1/2 dans des cellules lors de l���activation de la voie de signalisation MAPK. Par ailleurs, nous avons montr�� un nouveau domaine d���attache du module ERK1/2 sur Gab2. Des mutations sur les r��sidus essentiels de ce domaine d���attache n���entrainent pas seulement la dissociation de ERK1/2 avec Gab2, mais diminuent ��galement la phosphorylation d��pendante de ERK1/2 sur Gab2. Ainsi, nos donn��es montrent que la voie de signalisation MAPK r��gule les fonctions de la prot��ine Gab2 par le biais des kinases RSK et ERK1/2. Cette th��se ��labore par ailleurs un sch��ma complet des fonctions de Gab2 d��pendantes de la voie de signalisation MAPK dans le d��veloppement de nombreux cancers.

CXCR3 biased signaling, heteromerization and decoy properties

Le r��cepteur de chimiokine CXCR3 est un r��cepteur coupl�� �� la prot��ine G (RCPG) exprim��, entre autre, sur les cellules T activ��es lors d���une r��ponse immune. CXCR3 est activ�� par trois ligands inductibles par l���interf��ron-�� (CXCL9, 10, 11) et, plus r��cemment, il a ��t�� d��couvert que CXCL4 liait CXCR3. Nous savons que CXCR3 joue un r��le dans la chimiotaxie des leucocytes, mais peu d���attention a ��t�� port��e sur la signalisation biais��e induite par ces quatre ligands. Alors que l���homodim��risation entre r��cepteurs de chimiokine est un concept grandement observ��, l���h��t��rom��risation entre deux r��cepteurs reste un domaine de recherche active. La signalisation biais��e et l���h��t��rom��risation ont ��t�� test��es gr��ce �� la technique de bioluminescene resonance energy transfer (BRET) dans des cellules HEK293E. Nous pr��sentons une caract��risation pharmacologique des quatre ligands de CXCR3 et d��montrons l���h��t��rom��risation de CXCR3 avec CXCR4 et avec CXCR7. Nos r��sultats sugg��rent que les ligands de CXCR3 n���agissent pas de mani��re redondante.

Elucidation of the biological roles of Wnt5a signaling in follicle development

La sant�� folliculaire est d��termin��e par un nombre de facteurs endocriniens, paracrines et autocrines. Les gonadotrophines hypophysaires sont les principaux moteurs du d��veloppement du follicule, mais leurs actions sont modul��es localement par les hormones et des facteurs de croissance. Les glycoprot��ines de la famille des WNTs repr��sentent une grande famille de mol��cules impliqu��es dans diff��rentes voies de signalisation. Ils sont s��cr��t��s dans le but de moduler et coordonner la r��ponse des follicules aux gonadotrophines, et leurs activit��s sont indispensables �� la fonction ovarienne et �� la fertilit�� f��minine. Les WNTs sont g��n��ralement class��s en fonction de la (des) voie(s) qu���ils activent. Le r��le des membres de la voie canonique WNT et de ses composants tels que CTNNB1, WNT4, WNT2, FZD1 et FZD4 est bien ��tabli au cours du d��veloppement du follicule chez les rongeurs. Un r��le similaire des WNTs dans les esp��ces mono-ovulatoires demeure essentiellement inconnu. De plus, le r��le des WNT non canoniques dans l'ovaire de rongeurs est m��connu. Les objectifs de cette th��se sont (1) d'��lucider la r��gulation hormonale de l'expression de WNT5A et le r��le physiologique de WNT5A dans les cellules de la granulosa bovine in vitro et (2) d'identifier les r��les physiologiques de WNT5A dans l'ovaire de souris par inactivation g��nique conditionnelle. Chacun de ces objectifs a men�� �� la publication d���un article �� partir des r��sultats obtenus au cours de cette th��se. Dans le premier article, le r��le de WNT5A dans les cellules de la granulosa bovine a ��t�� ��tudi�� in vitro. Nous avons constat�� que WNT5A est un r��gulateur n��gatif de la st��ro��dogen��se stimul��e par la FSH issue des cellules de la granulosa, et qu'il agit en supprimant l'activit�� de signalisation des WNTs canoniques tout en induisant la voie de signalisation MAPK8/JUN. le deuxi��me article, afin d���examiner le r��le de deux WNTs non-canoniques, WNT5A et WNT11, �� diff��rents stades de d��veloppement folliculaire, nous avons g��n��r�� des mod��les de souris knock-out conditionnels ciblant les cellules de la granulosa pour chacun de ces WNTs. Les r��sultats obtenus ont permis de mettre en ��vidence que WNT5A est n��cessaire pour assurer la fertilit�� normale chez la femelle, le d��veloppement folliculaire et la st��ro��dogen��se ovarienne. Il est aussi un antagoniste de la r��ponse aux gonadotrophines, agissant par l���interm��diaire de la suppression de la signalisation canonique des WNTs. Chez les souris knock-out pour WNT11, nous ne constatons aucun d��faut important dans la fertilit�� des femelles. L���ensemble de notre travail met en ��vidence que WNT5A est essentiel pour le d��veloppement normal du follicule et qu���il agit pour inhiber la diff��renciation des cellules de la granulosa. En r��sum��, nous avons fourni une ��tude novatrice et approfondie, utilisant plusieurs mod��les et techniques pour d��terminer les m��canismes par lesquels WNT5A r��gule le d��veloppement des follicules.

Dopamine D1 and D2 Receptor Gene Expression and cGMP, IP3 and Ca2+ Regulation in the Brain Regions of Hypoxic Neonatal Rats: Role of Glucose, Oxygen and Epinephrine Supplementation

Department of Biotechnology, Cochin University of Science and Technology

Unveiling the components of the signaling pathway between endosomes and the phagocytic uptake machinery in Dictyostelium discoideum

The soil amoebae Dictyostelium discoideum take up particles from their environment in order to obtain nutrition. The particle transits through the cell within a phagosome that fuses with organelles of different molecular compositions, undergoing a gradual degradation by different sets of hydrolytic enzymes. Griffiths��� concept of ���phagosome individuality��� predicts signaling from phagosomes into the cytoplasm, which might regulate many aspects of cell physiology. The finding that Dictyostelium cells depleted of the lysozyme AlyA or over-expressing the esterase Gp70 exhibit increased uptake of food particles, led to the postulation of a signaling cascade between endocytic compartments and the cytoskeletal uptake machinery at the plasma membrane. Assuming that Gp70 acts downstream of AlyA, gene-expression profiling of both mutants revealed different and overlapping sets of misregulated genes that might participate in this signaling cascade. Based on these results, we analyzed the effects of the artificial misregulation of six candidate genes by over-expression or negative genetic interference, in order to reconstruct at least part of the signaling pathway. SSB420 and SSL793 were chosen as candidates for the first signaling step, as they were up-regulated in AlyA-null cells and remained unaltered in the Gp70 over-expressing cells. The over-expression of SSB420 enhanced phagocytosis and raised the expression levels of Gp70, supporting its involvement in the signaling pathway between AlyA and Gp70 as a positive regulator of phagocytosis. However, this was not the case of cells over-expressing SSL793, as this mutation had no effects on phagocytosis. For the signaling downstream of Gp70, we studied four commonly misregulated genes in AlyA-depleted and Gp70 over-expressing cells. The expression levels of SLB350, SSB389 and TipD were lower in both mutants and therefore these were assumed as possible candidates for the negative regulation of phagocytosis. Cells depleted of SLB350 exhibited an increased phagocytic activity and no effect on Gp70 expression, proving its participation in the signaling pathway downstream of Gp70. Unlike SLB350, the disruption of the genes coding for SSB389 and TipD had no effects on particle uptake, excluding them from the pathway. The fourth candidate was Yipf1, the only gene that was commonly up-regulated in both mutants. Yet, the artificial over-expression of this protein had no effects on phagocytosis, so this candidate is also not included in the signaling pathway. Furthermore, localizing the products of the candidate genes within the cell helped unveiling several cellular organelles that receive signals from the phagosome and transduce them towards the uptake machinery.

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41��, sap190��, ppm1��, rrd1��) and U34 modification defects (elp3��, kti12��, urm1��, ncs2��) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3�� deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3�� cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3��, urm1��) enhances nuclear import of and NCR gene activation (MEP2, GAP1) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TORsensitive NCR branch via Gln3 misregulation.

Differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis in brown adipocytes

Considering the major role of insulin signaling on fatty acid synthesis via stimulation of lipogenic enzymes, differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis have been investigated by comparing the individual lipogenic fluxes in WT and IRS-1 knockout (IRS-1 KO) brown adipocytes. Results from experiments on WT and IRS-1 KO cells incubated with [5-����C] glutamine were consistent with the existence of reductive carboxylation pathway. Analysis of isotopomer distribution of nine metabolites related to the lipogenic routes from glucose and glutamine in IRS-1 KO cells using [U-����C] glutamine as compared to that in WT cells indicated that flux through reductive carboxylation pathway was diminished while flux through conventional TCA cycle was stimulated due to absence of insulin signaling in IRS-1 KO cells. This observation was confirmed by quantitative estimation of individual lipogenic fluxes in IRS-1 KO cells and their comparison with fluxes in WT cells. Thus, these results suggest that glutamine���s substantial contribution to fatty acid synthesis can be directly manipulated by controlling the flux through reductive carboxylation of alpha-ketoglutarate to citrate using hormone (insulin).

"Functional Upregulation of Ca2+ -Activated K+ Channels in the Development of Substantia Nigra Dopamine Neurons"

Many connections in the basal ganglia are made around birth when animals are exposed to a host of new affective, cognitive, and sensori-motor stimuli. It is thought that dopamine modulates cortico-striatal synapses that result in the strengthening of those connections that lead to desired outcomes. We propose that there must be a time before which stimuli cannot be processed into functional connections, otherwise it would imply an effective link between stimulus, response, and reward in uterus. Consistent with these ideas, we present evidence that early in development dopamine neurons are electrically immature and do not produce high-frequency firing in response to salient stimuli. We ask first, what makes dopamine neurons immature? and second, what are the implications of this immaturity for the basal ganglia? As an answer to the first question, we find that at birth the outward current is small (3nS-V), insensitive to Ca2z, TEA, BK, and SK blockers. Rapidly after birth, the outward current increases to 15nS-V and becomes sensitive to Ca2z, TEA, BK, and SK blockers. We make a detailed analysis of the kinetics of the components of the outward currents and produce a model for BK and SK channels that we use to reproduce the outward current, and to infer the geometrical arrangement of BK and Ca2z channels in clusters. In the first cluster, T-type Ca2z and BK channels are coupled within distances of *20 nm (200 A��). The second cluster consists of L-type Ca2z and BK channels that are spread over distances of at least 60 nm. As for the second question, we propose that early in development, the mechanism of action selection is in a ������locked-in������ state that would prevent dopamine neurons from reinforcing cortico-striatal synapses that do not have a functional experiential- based value.

Functional Upregulation of Ca2+ -Activated K+ Channels in the Development of Substantia Nigra Dopamine Neurons

Many connections in the basal ganglia are made around birth when animals are exposed to a host of new affective, cognitive, and sensori-motor stimuli. It is thought that dopamine modulates cortico-striatal synapses that result in the strengthening of those connections that lead to desired outcomes. We propose that there must be a time before which stimuli cannot be processed into functional connections, otherwise it would imply an effective link between stimulus, response, and reward in uterus. Consistent with these ideas, we present evidence that early in development dopamine neurons are electrically immature and do not produce high-frequency firing in response to salient stimuli. We ask first, what makes dopamine neurons immature? and second, what are the implications of this immaturity for the basal ganglia? As an answer to the first question, we find that at birth the outward current is small (3nS-V), insensitive to Ca2+, TEA, BK, and SK blockers. Rapidly after birth, the outward current increases to 15nS-V and becomes sensitive to Ca2+, TEA, BK, and SK blockers. We make a detailed analysis of the kinetics of the components of the outward currents and produce a model for BK and SK channels that we use to reproduce the outward current, and to infer the geometrical arrangement of BK and Ca2+ channels in clusters. In the first cluster, T-type Ca2+ and BK channels are coupled within distances of similar to 20 nm (200 parallel to). The second cluster consists of L-type Ca2+ and BK channels that are spread over distances of at least 60 nm. As for the second question, we propose that early in development, the mechanism of action selection is in a "locked-in" state that would prevent dopamine neurons from reinforcing cortico-striatal synapses that do not have a functional experiential-based value.

Suburban immigrants to wildlands disrupt honest signaling in ultra-violet plumage

Urbanization changes habitat in a multitude of ways, including altering food availability. Access to human-provided food can change the relationship between body condition and honest advertisements of fitness, which may result in changes to behavior, demography, and metapopulation dynamics. We compared plumage color, its relationship with body condition and feather growth, and use as signal of dominance between a suburban and a wildland population of Florida Scrub-Jay (Aphelocoma coerulescens). Although plumage color was not related to body condition at either site, suburban birds had plumage with a greater proportion of total reflectance in the ultra-violet (UV) and peak reflectance at shorter wavelengths. Despite the use of plumage reflectance as a signal of dominance among individuals in the wildlands, we found no evidence of status signaling at the suburban site. However, birds emigrating from the suburban site to the wildland site tended to be more successful at acquiring breeder status but less successful at reproducing than were immigrants from an adjacent wildland site, suggesting that signaled and realized quality differ. These differences in signaling content among populations could have demographic effects at metapopulation scales and may represent an evolutionary trap whereby suburban immigrants are preferred as mates even though their reproductive success relative to effort is lower.

Effects of neuropathy on high-voltage-activated Ca2+ current in sensory neurones

Voltage-dependent Ca2+ channels (VDCCs) have emerged as targets to treat neuropathic pain; however, amongst VDCCs, the precise role of the CaV2.3 subtype in nociception remains unproven. Here, we investigate the effects of partial sciatic nerve ligation (PSNL) on Ca2+ currents in small/medium diameter dorsal root ganglia (DRG) neurones isolated from CaV2.3(���/���) knock-out and wild-type (WT) mice. DRG neurones from CaV2.3(���/���) mice had significantly reduced sensitivity to SNX-482 versusWTmice. DRGs from CaV2.3(���/���) mice also had increased sensitivity to the CaV2.2 VDCC blocker -conotoxin. In WT mice, PSNL caused a significant increase in -conotoxin-sensitivity and a reduction in SNX-482-sensitivity. In CaV2.3(���/���) mice, PSNL caused a significant reduction in -conotoxin-sensitivity and an increase in nifedipine sensitivity. PSNL-induced changes in Ca2+ current were not accompanied by effects on voltagedependence of activation in either CaV2.3(���/���) or WT mice. These data suggest that CaV2.3 subunits contribute, but do not fully underlie, drug-resistant (R-type) Ca2+ current in these cells. In WT mice, PSNL caused adaptive changes in CaV2.2- and CaV2.3-mediated Ca2+ currents, supporting roles for these VDCCs in nociception during neuropathy. In CaV2.3(���/���) mice, PSNL-induced changes in CaV1 and CaV2.2 Ca2+ current, consistent with alternative adaptive mechanisms occurring in the absence of CaV2.3 subunits.

Short communication: Suppressor of cytokine signaling-2 mRNA increases after parturition in the liver of dairy cows

After parturition, the somatotropic axis of the dairy cow is uncoupled, partly because of reduced concentration of liver-specific GH receptor (GHR) 1A. Estradiol-17 beta (E-2) concentrations increase at parturition and E-2 upregulates suppressors of cytokine signaling-2 (SOCS-2) mRNA expression, potentially inhibiting GH signaling. Therefore, we hypothesized that SOCS-2 mRNA is upregulated after parturition. Multiparous Holstein cows (n = 18) were dried off 45 d before expected parturition and fed diets to meet nutrient requirements at ad libitum or limited dry matter intake during the dry period. All cows were fed the same diet ad libitum from calving until 4 wk after parturition. Blood samples were collected weekly and more frequently near parturition. Liver biopsies obtained at -21, -7, 2, and 28 d relative to parturition were assessed for SOCS-2 and GHR 1A mRNA by quantitative real-time reverse-transcription PCR. The relative amount of SOCS-2 mRNA increased after parturition with both treatments and was greater on d 2 for cows limit-fed during the dry period compared with cows fed at ad libitum dry matter intake. Plasma E2 concentrations increased on d -13, -5 and 1 relative to parturition and the increases were greater in limit-fed cows. Plasma GH concentration was greater for limit-fed cows and increased after parturition in all cows. The amount of GHR 1A mRNA did not differ between diets but decreased on d 2. In addition to reduced GHR 1A, increased SOCS-2 mRNA after parturition, perhaps because of increased E-2, may further uncouple GH signaling in the liver of the transition dairy cow.