49 research outputs found
A Microinjectable Biological System, the Xenopus Oocyte, as an Approach to Understanding Signal Transduction Protein Function
No full textFibroblast growth factors 1 and 2 differently activate MAP kinase in Xenopus oocytes expressing fibroblast growth factor receptors 1 and 4
No full textAbstractThe mitogen-activated protein kinase (MAP kinase) signalling cascade activated by fibroblast growth factors (FGF1 and FGF2) was analysed in a model system, Xenopus oocytes, expressing fibroblast growth factor receptors (FGFR1 and FGFR4). Stimulation of FGFR1 by FGF1 or FGF2 and FGFR4 by FGF1 induced a sustained phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) and meiosis reinitiation. In contrast, FGFR4 stimulation by FGF2 induced an early transient activation of ERK2 and no meiosis reinitiation. FGFR4 transduction cascades were differently activated by FGF1 and FGF2. Early phosphorylation of ERK2 was blocked by the dominant negative form of growth factor-bound protein 2 (Grb2) and Ras, for FGF1–FGFR4 and FGF2–FGFR4. The phosphatidylinositol 3-kinase (PI3 kinase) inhibitors wortmannin and LY294002 only prevented the early ERK2 phosphorylation triggered by FGF2–FGFR4 but not by FGF1–FGFR4. ERK2 phosphorylation triggered by FGFR4 depended on the Grb2/Ras pathway and also involved PI3 kinase in a time-dependent manner
RasGAP is involved in signal transduction triggered by FGF1 in Xenopus oocytes expressing FGFR1
No full textAbstractThe role of RasGAP was investigated in the model system of Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) stimulated by fibroblast growth factor 1 (FGF1). The injection of the SH2-SH3-SH2 domains of RasGAP suppressed Ras activity, extracellular signal-regulated protein kinase 2 (ERK2) phosphorylation and Mos synthesis. The SH2 domain of Src, and PP2, an inhibitor of Src, also abolished Ras activity, ERK2 phosphorylation and Mos synthesis. In addition, Src activity was blocked by the SH2-SH3-SH2 domains of RasGAP. Immunoprecipitation of a chimera composed of the extracellular domain of the platelet-derived growth factor (PDGF) receptor and the intracellular domain of FGFR1 stimulated by PDGF-BB demonstrates the recruitment of phosphorylated RasGAP. This study shows that the transduction cascade induced by the FGFR1–FGF1 interaction in Xenopus oocytes involves RasGAP as a co-activator of Src to stimulate the Ras/mitogen-activated protein kinase cascade and Mos synthesis. It emphasises a new positive regulatory role for RasGAP in FGFR transduction
Fibroblast and epidermal growth factor receptor expression in Xenopus oocytes displays distinct calcium oscillatory patterns
No full textAbstractElectrophysiological study performed with the voltage clamp technique was used to examine the intracellular calcium pathway activated by tyrosine kinase receptor members. Three FGF receptors from Pleurodeles PR1, PR3, PR4, homologs to human receptors, and the human EGF receptor were expressed in Xenopus oocytes. Under FGF1, FGF2 and FGF4 stimulation, PR1 and PR3 display a one phase inward chloride calcium dependent current superimposed by sustained oscillations, whereas PR4 did not show any oscillations. These currents were dependent on intracellular calcium mobilisation, as the responses were reduced by caffeine (10 mM). Solely PR4 responses were affected by an extracellular calcium depleted solution suggesting the involvement of concomitant extracellular and intracellular calcium intervention in the calcium chloride current, whereas PR1 and PR3 did not. Under EGF stimulation, the EGF receptor elicits a two component inward current composed of an undelayed rapid transient dependent on intracellular calcium store recruitment followed by a second slower current dependent on calcium influx. The specific pattern and amplitude of the calcium oscillations induced by the combinatorial action of growth factors on their receptors could be relevant in numerous calcium dependent cell functions
ERK2 is required for FGF1-induced JNK1 phosphorylation in Xenopus oocyte expressing FGF receptor 1
No full textAbstractA possible connection between the ERK2 and JNK1 MAP kinases transduction cascades was investigated in Xenopus oocytes expressing FGFR1 stimulated by FGF1. Injection of various inhibitors for the Shc/Grb2/Ras/Mos/MEK/ERK2 cascade blocked FGF1-induced germinal vesicle breakdown (GVBD), as well as ERK2 and JNK1 phosphorylation. JNK1 was found to be activated downstream of ERK2, since injection of an active ERK2 triggered JNK1 phosphorylation and inhibition of ERK2 either by a MEK inhibitor or the MKP3 phosphatase blocked JNK1 phosphorylation. These results demonstrated that in FGFR1 signalling JNK1 phosphorylation depends on ERK2
Transduction cascades initiated by fibroblast growth factor 1 on Xenopus oocytes expressing MDA-MB-231 mRNAs
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A Toxoplasma gondii leucine-rich repeat protein binds phosphatase type 1 protein and negatively regulates its activity.
No full textWe have characterized the Toxoplasma gondii protein phosphatase type 1 (TgPP1) and a potential regulatory binding protein belonging to the leucine-rich repeat protein family, designated TgLRR1. TgLRR1 is capable of binding to TgPP1 to inhibit its activity and to override a G(2)/M cell cycle checkpoint in Xenopus oocytes. In the parasite, TgLRR1 mRNA and protein are both highly expressed in the rapidly replicating and virulent tachyzoites, while only low levels are detected in the slowly dividing and quiescent bradyzoites. TgPP1 mRNA and protein levels are equally abundant in tachyzoites and bradyzoites. Affinity pull down and immunoprecipitation experiments reveal that the TgLRR1-TgPP1 interaction takes place in the nuclear subcompartment of tachyzoites. These results are consistent with those of localization studies using both indirect immunofluorescence with specific polyclonal antibody and transient transfection of T. gondii vector expressing TgLRR1 and TgPP1. The inability to obtain stable transgenic tachyzoites suggested that overexpression of TgLRR1 and TgPP1 may impair the parasite's growth. Together with the activation of Xenopus oocyte meiosis reinitiation, these data indicate that TgLRR1 protein could play a role in the regulation of the T. gondii cell cycle through the modulation of phosphatase activity.info:eu-repo/semantics/publishe
Schistosoma mansoni: Structural and biochemical characterization of two distinct Venus Kinase Receptors
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