3-phosphoinositide-dependent protein kinase 1 (PDK1) mediates crosstalk between Src and Akt pathways in MET receptor signaling.

The high-affinity tyrosine kinase receptor MET plays a pivotal role in several facets of cell regulation. Although its mitogenic effect is well documented, some aspects of connection patterns between signaling pathways involved in cell cycle progression remain to be deciphered. We have used a tractable heterologous expression system, the Xenopus oocyte, to detect connections between distinct MET signaling cascades involved in G2/M progression. Our results reveal that Src acts as an adapter via its SH2 domain to recruit 3-phosphoinositide-dependent protein kinase 1 (PDK1) to the MET signaling complex leading to Akt phosphorylation. These data define an original crosstalk between Src and Akt signaling pathways that contributes to MET-induced entry into the M phase, and deserves further investigation in pathologies harboring deregulation of this receptor.

Effects of glyphosate and a commercial formulation Roundup® exposures on maturation of Xenopus laevis oocytes.

Pesticides are often found at high concentrations in small ponds near agricultural field where amphibians are used to live and reproduce. Even if there are many studies on the impacts of phytopharmaceutical active ingredients in amphibian toxicology, only a few are interested in the earlier steps of their life cycle. While their populations are highly threatened with extinction. The aim of this work is to characterize the effects of glyphosate and its commercial formulation Roundup® GT Max on the Xenopus laevis oocyte maturation which is an essential preparation for the laying and the fertilization. Glyphosate is an extensively used herbicide, not only known for its effectiveness but also for its indirect impacts on non-target organisms. Our results showed that exposures to both forms of glyphosate delayed this hormone-dependent process and were responsible for spontaneous maturation. Severe and particular morphogenesis abnormalities of the meiotic spindle were also observed. The MAPK pathway and the MPF did not seem to be affected by exposures. The xenopus oocyte is particularly affected by the exposures and appears as a relevant model for assessing the effects of environmental contamination.

Adverse effects of fly ashes used as immobilizing agents for highly metal-contaminated soils on Xenopus laevis oocytes survival and maturation-a study performed in the north of France with field soil extracts.

Amphibians are now recognized as the most endangered group. One of this decline causes is the degradation of their habitat through direct contamination of water, soil leaching, or runoff from surrounding contaminated soils and environments. In the North of France, the extensive industrial activities resulted in massive soil contamination by metal compounds. Mineral amendments were added to soils to decrease trace metal mobility. Because of the large areas to be treated, the use of inexpensive industrial by-products was favored. Two types of fly ashes were both tested in an experimental site with the plantation of trees in 2000. Aim of the present work was to investigate the effects of extracts from metal-contaminated soils treated or not for 10 years with fly ashes on Xenopus laevis oocyte using cell biology approaches. Indeed, our previous studies have shown that the Xenopus oocyte is a relevant model to study the metal ion toxicity. Survival and maturation of oocyte exposed to the soil extracts were evaluated by phenotypic approaches and electrophysiological recordings. An extract derived from a metal-contaminated soil treated for 10 years with sulfo-calcic ashes induced the largest effects. Membrane integrity appeared affected and ion fluxes in exposed oocytes were changed. Thus, it appeared that extracted elements from certain mineral amendments used to prevent the mobility of metals in the case of highly metal-contaminated soils could have a negative impact on X. laevis oocytes.

Maturation of Xenopus laevis oocytes under cadmium and lead exposures: Cell biology investigations.

Since amphibians are recognised as good models to assess the quality of environments, only few studies have dealt with the impacts of chemical contaminants on their gametes, while toxic effects at this stage will alter all the next steps of their life cycle. Therefore, we propose to investigate the oocyte maturation of Xenopus laevis in cadmium- and lead-contaminated conditions. The impacts of cadmium and lead ions were explored on events involved in the hormone-dependent process of maturation. In time-course experiments, cadmium, at the highest concentration, delayed and prevented the germinal vesicle breakdown. Even in the absence of progesterone this ion could also induce it. No such spontaneous maturation was observed after lead exposures. An acceleration of the process at the highest tested concentration of lead (90µM), in presence of progesterone, was recorded. Cytological observations highlighted that cadmium exposures drove severe disturbances of meiotic spindle morphogenesis. At last, cadmium exposures altered the MAPK pathway, regarding the activation of ERK2 and RSK, but also the activation and the activity of the MPF, by disturbing the state of phosphorylation of Cdc2 and histone H3. Xenopus laevis oocytes were affected by these metal ion exposures, notably by Cd2+. Signatures of these metal exposures on the oocyte maturation were detected. This germ cell appeared to be a relevant model to assess the effects of environmental contaminants such as metals.

Cadmium but not lead exposure affects Xenopus laevis fertilization and embryo cleavage.

Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3.

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G2/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G2/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G2/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.

Insulin-induced cell division is controlled by the adaptor Grb14 in a Chfr-dependent manner.

Beyond its key role in the control of energy metabolism, insulin is also an important regulator of cell division and neoplasia. However, the molecular events involved in insulin-driven cell proliferation are not fully elucidated. Here, we show that the ubiquitin ligase Chfr, a checkpoint protein involved in G2/M transition, is a new effector involved in the control of insulin-induced cell proliferation. Chfr is identified as a partner of the molecular adapter Grb14, an inhibitor of insulin signalling. Using mammalian cell lines and the Xenopus oocyte as a model of G2/M transition, we demonstrate that Chfr potentiates the inhibitory effect of Grb14 on insulin-induced cell division. Insulin stimulates Chfr binding to the T220 residue of Grb14. Both Chfr binding site and Grb14 C-ter BPS-SH2 domain, mediating IR binding and inhibition, are required to prevent insulin-induced cell division. Targeted mutagenesis revealed that Chfr ligase activity and phosphorylation of its T39 residue, a target of Akt, are required to potentiate Grb14 inhibitory activity. In the presence of insulin, the binding of Chfr to Grb14 activates its ligase activity, leading to Aurora A and Polo-like kinase degradation and blocking cell division. Collectively, our results show that Chfr and Grb14 collaborate in a negative feedback loop controlling insulin-stimulated cell division.

Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis.

Protein kinases (PKs) are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt) which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae.

SmSak, the second Polo-like kinase of the helminth parasite Schistosoma mansoni: conserved and unexpected roles in meiosis.

Polo-like kinases (Plks) are a family of conserved regulators of a variety of events throughout the cell cycle, expanded from one Plk in yeast to five Plks in mammals (Plk1-5). Plk1 is the best characterized member of the Plk family, homolog to the founding member Polo of Drosophila, and plays a major role in cell cycle progression by triggering G2/M transition. Plk4/Sak (for Snk (Serum-inducible kinase) akin kinase) is a unique member of the family, structurally distinct from other Plk members, with essential functions in centriole duplication. The genome of the trematode parasite Schistosoma mansoni contains only two Plk genes encoding SmPlk1 and SmSak. SmPlk1 has been shown already to be required for gametogenesis and parasite reproduction. In this work, in situ hybridization indicated that the structurally conserved Plk4 protein, SmSak, was largely expressed in schistosome female ovary and vitellarium. Expression of SmSak in Xenopus oocytes confirmed its Plk4 conserved function in centriole amplification. Moreover, analysis of the function of SmSak in meiosis progression of G2-blocked Xenopus oocytes indicated that, in contrast to SmPlk1, SmSak cannot induce G2/M transition in the absence of endogenous Plk1 (Plx1). Unexpectedly, meiosis progression was spontaneously observed in Plx1-depleted oocytes co-expressing SmSak and SmPlk1. Molecular interaction between SmSak and SmPlk1 was confirmed by co-immunoprecipitation of both proteins. These data indicate that Plk1 and Plk4 proteins have the potential to interact and cross-activate in cells, thus attributing for the first time a potential role of Plk4 proteins in meiosis/mitosis entry. This unexpected role of SmSak in meiosis could be relevant to further consider the function of this novel Plk in schistosome reproduction.

Schistosoma mansoni: structural and biochemical characterization of two distinct Venus Kinase Receptors.

Venus Kinase Receptors (VKRs) are atypical transmembrane proteins composed of an extracellular Venus FlyTrap module linked through a single helix to a tyrosine kinase domain similar to that of insulin receptors. This structure was first described in Schistosoma mansoni, then in a selected range of invertebrates, including many insects. The preferential expression of VKRs in larvae and gonads suggested their role in development and reproduction. While a single vkr gene was consistently found in all genomes, we identified two distinct vkr genes in S. mansoni. Our data indicated that Smvkr1 and Smvkr2 are very similar in structure and likely originated from gene duplication. Both genes are expressed in all the parasite stages and encode homologous proteins with a conserved VKR structure. Recombinant SmVKR1 and SmVKR2 exhibit tyrosine kinase activities dependent on the binding of distinct small ligand molecules. SmVKR1 and SmVKR2 could represent paralogs with different functions in the parasite.

Akt interaction with PLC(gamma) regulates the G(2)/M transition triggered by FGF receptors from MDA-MB-231 breast cancer cells.

BACKGROUND/AIM: Estrogen-independent breast cancer cell growth is under the control of fibroblast growth factors receptors (FGFRs), but the role of phospholipase C gamma (PLC(gamma)) and Akt, the downstream effectors activated by FGFRs, in cell proliferation is still unresolved. MATERIALS AND METHODS: FGFRs from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a powerful model system to assess the G(2)/M checkpoint regulation. Under FGF1 stimulation, an analysis of the progression in the M-phase of the cell cycle and of the Akt signaling cascades were performed using the phosphatidylinositol-3-kinase inhibitor, LY294002, and a mimetic peptide of the SH3 domain of PLC(gamma). RESULTS: Activated Akt binds and phosphorylates PLC(gamma) before Akt targets the tumor suppressor Chfr. Disruption of the Akt-PLC(gamma) interaction directs Akt binding to Chfr and accelerates the alleviation of the G(2)/M checkpoint. CONCLUSION: The PLC(gamma)-Akt interaction, triggered by FGF receptors from estrogen-independent breast cancer cells MDA-MB-231, regulates progression in the M-phase of the cell cycle.

NMR observation of Tau in Xenopus oocytes.

The observation by NMR spectroscopy of microinjected 15N-labelled proteins into Xenopus laevis oocytes might open the way to link structural and cellular biology. We show here that embedding the oocytes into a 20% Ficoll solution maintains their structural integrity over extended periods of time, allowing for the detection of nearly physiological protein concentrations. We use these novel conditions to study the neuronal Tau protein inside the oocytes. Spectral reproducibility and careful comparison of the spectra of Tau before and after cell homogenization is presented. When injecting Tau protein into immature oocytes, we show that both its microtubule association and different phosphorylation events can be detected.

A non-canonical Grb2-PLC-gamma1-Sos cascade triggered by lipovitellin 1, an apolipoprotein B homologue.

The injection of the Grb2 adapter in Xenopus oocytes promotes G2/M transition without stimulation from a receptor only the first day after the oocytes removal from the ovaries. This cell cycle reinitiation is Ras-dependent and requires the SH2 and SH3 domains of Grb2. The SH2 domain of Grb2 binds the tyrosine phosphorylated lipovitellin1, a homologue of the human apolipoprotein B. The N-SH3 domain of Grb2 is linked to a proline-rich sequence of the C2 domain of PLC-gamma1, PLC-gamma1 itself is linked, through its SH3 domain, to the C-terminal proline-rich region of Sos. When Grb2-PLC-gamma1-Sos is associated, PLC-gamma1 is not phosphorylated on Y783 but shows a phospholipase activity. Inhibition of lipovitellin 1 or PLC-gamma1 avoids Grb2-induced cell cycle reinitiation. Therefore, the Grb2-lipovitellin 1 association is the starting point of a novel signaling pathway, where PLC-gamma1 binds Grb2 and recruits Sos.

FGF receptor phosphotyrosine 766 is a target for Grb14 to inhibit MDA-MB-231 human breast cancer cell signaling.

BACKGROUND: Fibroblast growth factors receptors (FGFRs) are involved in estrogen-independent breast cancer cell growth. Grbl4, a member of the Grb7 family of adapters, is an inhibitor of FGFR signaling. MATERIALS AND METHODS: FGFR from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a widely used model system to question cascade transduction regulations. The effect of microinjection of Grb14 and various mimetic peptides for FGFR tyrosine residues were analysed by FGFR immunoprecipitation and Western blot analysis of signaling cascades. RESULTS: PLCy, ERK2, JNK1 and AKT were blocked by Grb14. Only the pY766 phosphopeptide mimetic of the PLCgamma binding site on FGFR released the inhibitory action of Grb14. CONCLUSION: Grb14 binds to the Y766 site of MDA-MB-231-FGFR, competing for PLCy activation, thus inducing an arrest of the signaling transduction cascades.