Bub1 is activated by the protein kinase p90Rsk during Xenopus oocyte maturation

AbstractBackground: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90Rsk, which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90Rsk involves components of the spindle assembly checkpoint.Results: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90Rsk, restores the activation of Bub1 in the presence of U0126. Moreover, purified p90Rsk phosphorylates Bub1 in vitro and increases its protein kinase activity.Conclusions: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90Rsk, is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90Rsk and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90Rsk

0107: Strategy of early detection and active management of supraventricular arrhythmia with remote monitoring: the randomized, multicenter SETAM trial

ObjectiveAtrial fibrillation (AF) is a common arrhythmia associated with increased risk of thromboembolic events or other complications. The French randomized, multicenter, SETAM trial assessed the impact of the home monitoring (HM) technology on detection and treatment of supra-ventricular arrhythmia (SVA).MethodsPatients (pts) implanted with a dual chamber pacemaker were enrolled in the study at hospital discharge if they had a sinusal rhythm at enrollment, no antiarrhythmic, anticoagulant or dual-antiplatelet therapy, and if they had a CHA2DS2-VASc score of 2 or more. The pts were randomly assigned to an active group (Act Gp), followed by Biotronik HM, or a control group (Cont Gp) without HM surveillance. The time from implantation to the first SVA-related intervention was compared between the 2 groups (primary endpoint).ResultsA total of 595 pts (mean age = 79±8 y.o, 63% male, mean CHA2DS2-VASc score = 3.7±1.2) were followed during 12.8±3.3Mo. The most prevalent co-morbidities were hypertension (82% pts), diabetes (29%) and vascular disease (24%). Implantation indications were atrio-ventricular blocks in 77% of pts, sinus node disease in 20% and others in 3%.The global SVA incidence was 25% (29% in the Act Gp vs 22% in the Cont Gp, p=ns).A therapy (drugs or ablation) was instituted for 49/291 pts (17%) in the Act Gp vs 43/304 pts (14%) in the Cont Gp (p=ns). The median time from implantation to the first therapy for SVA was 114 [44; 241] days in the Act Gp vs 224 [67; 366] days in the Cont Gp, representing a median gain of 110-days in SVA management (50% reduction, p=0.01). Over these 92 pts, 54 had AF (59%) and 38 had atrial flutter or tachyarrhythmia (41%). Anticoagulation was initiated in 80% of pts and antiarrhythmic drugs in 55%.ConclusionThe SETAM study demonstrated that HM allows earlier detection and treatment of SVA in pacemaker pts. The next step is to report how early detection of SVA with HM can possibly improve the patients clinical outcome

Cycle Inhibiting Factors (Cifs): Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells

Cycle inhibiting factors (Cifs) are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL) complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions

Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria

Détermination de la voie de signalisation cellulaire eucaryote détournée par la protéine bactérienne Cif

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

The Enteropathogenic Escherichia coli Effector Cif Induces Delayed Apoptosis in Epithelial Cells▿

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins, the cyclomodulins, which modulate the host cell cycle. Upon injection into the host cell by the type III secretion system of enteropathogenic Escherichia coli (EPEC), Cif induces both G2 and G1 cell cycle arrests. The cell cycle arrests correlate with the accumulation of p21waf1 and p27kip1 proteins that inhibit CDK-cyclin complexes, whose activation is required for G1/S and G2/M transitions. Increases of p21 and p27 levels are independent of p53 transcriptional induction and result from protein stabilization through inhibition of the ubiquitin/proteasome degradation pathway. In this study, we show that Cif not only induces cell cycle arrest but also eventually provokes a delayed cell death. Indeed, 48 h after infection with EPEC expressing Cif, cultured IEC-6 intestinal cells were positive for extracellular binding of annexin V and exhibited high levels of cleaved caspase-3 and lactate dehydrogenase release, indicating evidence of apoptosis. Cif was necessary and sufficient for inducing this late apoptosis, and the cysteine residue of the catalytic site was required for Cif activity. These results highlight a more complex role of Cif than previously thought, as a cyclomodulin but also as an apoptosis inducer

Association of p34cdc2 kinase and MAP kinase with microtubules during the meiotic maturation of Xenopus oocytes.

International audiencep34cdc2 protein is found in prophase, metaphase and activated Xenopus oocytes at a similar level whereas its kinase activity oscillates within meiosis. Using an anti-PSTAIRE antibody that recognizes Xenopus p34cdc2, it was demonstrated that the major part of p34cdc2 was associated with microtubules isolated in vitro from Xenopus oocytes. Conversely, tubulin was recovered in association with p34cdc2 in p13-Sepharose pellets. The abundance of the fraction of p34cdc2 which was associated with microtubules did not oscillate during the meiotic maturation and the activation process. By contrast, the histone H1 kinase activity of p34cdc2 estimated in microtubular oocyte pellets was much higher in metaphase than in prophase oocytes. Cyclin B, which is associated in vivo with p34cdc2 in prophase and metaphase oocytes, was also present in the microtubular fractions. However, cyclin was not necessary for the binding of p34cdc2 to microtubules since p34cdc2 from activated eggs, where cyclin was missing, still copurified with microtubules. Purified MAP2, but not tubulin, was able to bind to p34cdc2, demonstrating that the association between p34cdc2 and microtubules was mediated by microtubule-associated proteins. During the meiotic maturation of Xenopus oocytes, several protein kinases were activated, among them MAP kinase. MAP kinase also associated with microtubules. It was demonstrated that both p34cdc2 kinase and MAP kinase purified from Xenopus oocytes were able to phosphorylate in vitro rat brain MAP2. However both protein kinases phosphorylated different domains of MAP2, suggesting that they might regulate microtubules in different ways

18F-fluorodihydroxyphenylalanine PET/CT in pheochromocytoma and paraganglioma: relation to genotype and amino acid transport system L

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Looking beyond the thyroid: advances in the understanding of pheochromocytoma and hyperparathyroidism phenotypes in MEN2 and of non-MEN2 familial forms

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